Methods controlling gene expression

ABSTRACT

The present invention is in the field of genetics, especially plant genetics, and provides agents capable of controlling gene expression. The present invention specifically provides sequences of naturally occurring, tissue-specifically expressed microRNAs. The invention further provides for transgenic expression constructs comprising sequences encoding such microRNAs. By incorporation of the microRNA encoding sequence the expression from the expression construct is specifically silenced in the tissue where the naturally occurring microRNA is naturally expressed. Thereby the expression profile resulting from the promoter is modulated and leakiness is reduced. The invention further provides for a method for modulating transgenic expression by incorporating sequences encoding the microRNAs into transgenic expression constructs. The compositions and methods of the invention can be used to enhance performance of agricultural relevant crops and for therapy, prophylaxis, research and diagnostics in diseases and disorders, which afflict mammalian species.

RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 11/918,554 filed Oct. 15, 2007, which is a national stageapplication (under 35 U.S.C. 371) of PCT/EP2006/061604 filed Apr. 13,2006, which claims benefit of U.S. application 60/672,976 filed Apr. 19,2005. The entire contents of each of these applications are herebyincorporated by reference herein in their entirety.

SUBMISSION OF SEQUENCE LISTING

The Sequence Listing associated with this application is filed inelectronic format via EFS-Web and hereby incorporated by reference intothe specification in its entirety. The name of the text file containingthe Sequence Listing is Sequence_Listing_074021_0072_01. The size of thetext file is 159 KB, and the text file was created on Dec. 4, 2017.

FIELD OF THE INVENTION

The present invention is in the field of genetics, especially plantgenetics, and provides agents capable of gene-specific silencing. Thepresent invention specifically provides polycicstronic RNA moleculescapable to generate double-stranded RNA (dsRNA) agents, methods forutilizing such molecules and cells and organism, especially plants,containing such molecules.

BACKGROUND OF THE INVENTION

Many factors affect gene expression in plants and other eukaryoticorganisms. Recently, small RNAs, 21-26 nucleotides, have emerged asimportant regulators of eukaryotic gene expression. The known smallregulatory RNAs fall into two basic classes. One class of small RNAs isthe short interfering RNAs (siRNAs). These play essential roles in RNAsilencing, a sequence-specific RNA degradation process that is triggeredby double-stranded RNA (dsRNA) (see Vance and Vaucheret (2001) Science292:2277-2280, and Zamore (2001) Nat Struct Biol 8:746-750 for recentreviews on RNA silencing in plants and animals, respectively). RNAsilencing plays a natural role in defense against foreign nucleic acidsincluding virus resistance in plants and control of transposons in anumber of organisms. siRNAs are double-stranded with small 3′ overhangsand derive from longer dsRNAs that induce silencing. They serve asguides to direct destruction of target RNAs and have been implicated asprimers in the amplification of dsRNA via the activity of a cellular RNAdependent RNA polymerase. In plants, si-like RNAs have also beenassociated with dsRNA-induced transcriptional gene silencing (TGS), aprocess in which dsRNA with homology to promoter regions triggers DNAmethylation and inhibits transcription. The TGS-associated small RNAs,unlike true siRNAs, are not involved in RNA degradation.

Another group of small RNAs are known generically as short temporal RNAs(stRNAs) and more broadly as micro-RNAs (miRNAs). miRNAs have emerged asevolutionarily conserved, RNA-based regulators of gene expression inanimals and plants. miRNAs (approx. 21 to 25 nt) arise from largerprecursors with a stem loop structure that are transcribed fromnon-protein-coding genes. miRNAs are single-stranded, and theiraccumulation is developmentally regulated and/or regulated byenvironmental stimuli. They derive from partially double-strandedprecursor RNAs that are transcribed from genes that do not encodeprotein. The miRNAs appear to be transcribed as hairpin RNA precursors,which are processed to their mature, about 21 nt forms by Dicer (Lee RD, and Ambros, V. Science 294: 862-864 (2001)). miRNA targets a specificmRNA to suppress gene expression at post-transcriptional level (i.e.degrades mRNA) or at translational level (i.e. inhibits proteinsynthesis). microRNAs (miRNAs) have emerged as evolutionarily conserved.There are several hundred of miRNAs have been recently identifiedthrough computational analysis and experimental approaches from manyplant and animal species. A body of miRNAs is well conserved withinplant kingdom or animal kingdom, but some are species or genus specific.

miRNA genes are first transcribed by Pol II RNA polymerase resulting inpri-miRNA with Cap structure at 5′ end and poly tail at 3′ end.Pri-miRNA is subjected to cleavage by an RNase III-like enzyme, Dicer,to generate mature miRNA. miRNA is then recruited into RISC (RNA inducedsilencing complex) and targets a specific mRNA in cytoplasm to suppressgene expression at post-transcriptional level (i.e. degrades mRNA).MiRNA can also inhibit protein synthesis after targeting a mRNA in asequence-specific manner. The mechanism of such translational inhibitionis to be uncovered. It has been shown both in animal and plant, pairingof the miRNA 5′ region to its target mRNA is crucial for miRNA actions(Mallory A et al., EMBO Journal 23:3356-3364, 2004; Doench J and SharpP, Genes & Development 504-511, (2004)).

Thus, it was realized that small, endogenously encoded hairpin RNAscould stably regulate gene expression via elements of the RNAimachinery. Like stRNAs (and unlike siRNAs involved in RNA silencing),most of the miRNAs lack complete complementarity to any putative targetmRNA. Although their functions are, as yet, not known, it ishypothesized that they regulate gene expression during development,perhaps at the level of development. However, given the vast numbers ofthese small regulatory RNAs, it is likely that they are functionallymore diverse and regulate gene expression at more than one level. Inplant, majority of miRNA target genes are transcription factors whichare required for meristem identity, cell division, organ separation, andorgan polarity. Some miRNAs have unique tissues-specific and/or temporalexpression pattern. McManus et al. (RNA 8:842-850 (2002)) also studiedmiRNA mimics containing 19 nucleotides of uninterrupted RNA duplex, a12-nucleotide loop length and one asymmetric stem-loop bulge composed ofa single uridine opposing a double uridine. Synthetic miRNA can eitherbe transfected into cells or expressed in the cell under the control ofan RNA polymerase III promoter and cause the decreased expression of aspecific target nucleotide sequence (McManus et al. (2002) RNA8:842-850, herein incorporated by reference).

In plant, there have been increasing evidences that microRNAs targetgenes involved in many aspects of plant growth and development such asmeristem identity, cell division, organ separation, and organ polarity.For example, miR164 targets NAC-domai genes, which encodes a family oftranscription factors including (CUP-SHAPED COTYLEDON1, CUC1 and CUC2).Expression of miR164-resistant version of CUC1 mRNA from the CUC1promoter causes alterations in Arabidopsis embryonic vegetative, andfloral development (Mallory A et al., Current Biology 14:1035-1046,(2004)). MiR166 mediates leaf polarity in Arabidopsis and maize (JuarezM et al., Nature 428: 84-88, (2004) and Kidner C and Martlenssen R,Nature 428: 81-84, (2004)). MiR172 directs flower development throughregulating APETALA2 gene expression (Chen X, Science, 303: 2022-2025(2004)). MiRNAs also regulate plant gene expression in response toenvironmental stimuli such as abiotic stress. For example, theexpression of miR395, the sulfurylase-targeting miRNA, is increased uponsulfate starvation (Jone-Rhoades M W and Bartel D, Molecular Cell 14:787-799, (2004)). MiR319c expression is upregulated by cold but notdehydration, NaCl or ABA (Sunkar R and Zhu J K, The Plant Cell16:2001:2019, (2004)). Some miRNAs have unique tissues-specific and/ortemporal expression patterns. For example, miR398b is expressedpredominantly in Arabidopsis leaf (Sunkar R and Zhu J K., The Plant Cell16:2001:2019, 2004)

In animals, miRNAs also play a key role in growth and development. Forexample, in mammals, miR181 modulates hematopietic lineagedifferentiation (Chen C Z et al., Science 303:83-86, (2004)), and MiR196direct cleavage of HOXB8 mRNA (Yekta S et al., Science 304:594-596,(2004)). In human, miR-124 is expressed only in brain with possible rolein neuronal differentiation (Sempere L. F. et al., Genome Biology 5:R13(2004)) while miR-1 is expressed in muscle (Lagos-Quintana. M et al.,Current Biology, (2002))

In plant, so far disclosed applications of miRNAs are

-   1) overexpression and/or ectopic expression of a given miRNA to    characterize its function or generate desired phenotypes (Palatnik J    et al., Nature 425: 257-263, (2003));-   2) engineering a miRNA precursor to produce new miRNA targeting    gene-of-interest (WO2004009779;-   3) engineering mRNA to be resistant to miRNA recognition and    cleavage (i.e. silent mutation—by changing nucleotides in the codons    for the same amino acid) (Palatnik J et al., Nature 425: 257-263,    2003; Mallory A et al., Current Biology 14:1035-1046, (2004)).

US 20040268441 describes microRNA precursor constructs that can bedesigned to modulate expression of any nucleotide sequence of interest,either an endogenous plant gene or alternatively a transgene.

One of the major obstacles in various field of biotechnology (includingbut not limited to gene therapy and plant biotechnology) is thedifficulty to achieve cell or tissue specificity. Transcription is anessential process for every living organism to convert abstract geneticinformation into physical reality. Promoter is a major component todrive transcription. Some promoters are active in every tissue (e.g.actin promoters) while other promoters only active in limited tissues.It is quite often that a given promoter is predominantly active in onetissue type but weakly expressed in some other tissues, so called leakypromoters. Those promoters are undesirable for agriculture andpharmaceutical application because unintended expression ofgene-of-interest resulted from leaky promoters could cause detrimentaleffects to crops or patients. It certainly would not meet requirement ofregulatory agency.

For example plant-parasitic nematodes cause diseases in all crops ofeconomic importance, resulting in an estimated US$100 billion annuallosses to world agriculture. In US, soybean cyst nematode is No. 1pest—infecting nearly all soybean production states (approx. 80 millionacres) and causes up to 30% yield loss each year. Chemical controlmeasures are inadequate and environmentally unfriendly. Transgenic-planttechnology offers a great potential, however, no significant success hasbeen made yet. One major problem is the leaky activities of nematodefeeding site ‘specific’ promoter. Although such promoter (e.g. TobRB7)could drive phytotoxic molecules to ‘kill’ the feeding cells andalleviate nematode infection, leaky expression of these phytotoxicmolecules in other tissues (e.g. flower) causes detrimental effects onthe host plants. Thus, a novel approach to control leaky expression isin high demand.

For example a major problem in chemotherapy and radiation therapy forcancer is the difficulty in achieving tumor-specific cell killing. Theinability of radiation or cytotoxic chemotherapeutic agents todistinguish between tumor cells and normal cells necessarily limits thedosage that can be applied. As a result, disease relapse due to residualsurviving tumor cells is frequently observed, and thus there exists aclear need for alternative non-surgical strategies. Development of genetherapy techniques is approaching clinical realization for the treatmentof neoplastic and metabolic diseases, and numerous genes displayinganti-tumor activity have been identified. However, the usefulness ofgene therapy methods has been limited due to systemic toxicity ofanti-tumor polypeptides encoded by gene therapy constructs (Spriggs &Yates (1992) in Bentler, ed., Tumor Necrosis Factor: The Molecules andTheir Emerging Roles in Medicine, pp. 383-406 Raven Press, New York,N.Y.; Sigel & Puri (1991) J Clin Oncol 9:694-704; Ryffel (1997)Immunopathol 83:18-20). Problems with current state-of-the-art genetherapy strategies include the inability to deliver the therapeutic genespecifically to the target cells. This leads to toxicity in cells thatare not the intended targets. For example, manipulation of-the p53 genesuppresses the growth of both tumor cells and normal cells, andintravenous administration of tumor necrosis factor alpha (TNF.alpha.)induces systemic toxicity with such clinical manifestations as fever andhypertension. Attempts have been made to overcome these problems. Theseinclude such strategies as the use of tissue-specific promoters to limitgene expression to specific tissues and the use of heat (Voellmy R., etal., Proc. Natl. Acad. Sci. USA, 82:4949-4953 (1985)) or ionizingradiation inducible enhancers and promoters (Trainman, R. H., et al.,Cell 46: 567-574 (1986); Prowess, R., et al., Proc. Natl. Acad. Sci. USA85, 7206-7210 (1988)) to enhance expression of the therapeutic gene in atemporally and spatially controlled manner.

Adenoviral vectors possess a number of attributes that render themuseful gene delivery vehicles for systemic gene therapy. Ideally, such asystem would be designed so that systemically administered vector wouldhome specifically to tumor target cells without ectopic infection ofnormal cells. However, a major stumbling block to this approach is thefact that the majority of adenoviral vectors administered systemicallyare sequestered in the liver. Therefore measures that specificallycontrol the distribution of delivered transgene expression must besuperimposed on the basic vector for optimal applicability of adenoviralvectors.

Unfortunately, for most of the presently expression systems expressionof the active ingrediant is not restricted to the tumor sites due to the‘leakiness’ of the available promoters thereby limiting efficiency ofsuch approaches. Tissue specific promoters may add a further degree oftransgene expression selectivity but there are few of these that havebeen validated in vivo and all are subject to some degree ofnon-specific activation or “leakiness”. A versatile mechanism forcontrollable gene expression is therefore highly desired for genetherapy.

A mechanism for controlling gene expression should ideally include bothspatial and temporal control of gene expression. One existing strategyemploys a chemically regulated signal, for example thetetracycline-inducible gene expression system (Gossen & Bujard (1992)Proc Natl Acad Sci USA 89:5547-5551; Gossen & Bujard (1993) Nuc AcidsRes 21(18):4411-4412; Gossen et al. (1995) Science 268:1766-1769). Asimilar approach involves the provision of ionizing radiation toactivate a radiosensitive promoter, e.g. the EGR-1 promoter(Weischelbaum et al. (1994) Cancer Res 54:4266-4269; Hallahan et al.(1995) Nat Med 1(8):786-791; Joki et al. (1995) Hum Gen Ther6:1507-1513). An alternative design relies on endogenous control of geneexpression. For example, the CEA promoter is selectively expressed incancer cells (Hauck & Stanners (1995) J Biol Chem 270:3602; Richards etal. (1995) Human Gene Ther 6:881-893).

In the past, several approaches have been attempted to solve leakinessproblem in plant gene expression without much success. By conducting aseries of deletion of promoter sequence, one might eliminate thesequence in the promoter region which contributes to the leakyexpression. For example, a deleted version of TbRB7 promoter drives GUSreporter gene expression in nematode feeding cells in the root uponnematode infection. Leaky expression, however, in flower tissue is stillunsolved (Opperman C H et al., Science 263:221-223, (1994)). By making achimeric promoter, i.e. a minimal promoter (e.g. 35S promoter) plustissues-specific regulatory elements, one might restrict gene expressionin desired tissues. However, if tissue-specific regulatory elements areleaky, the chimeric promoter will be leaky as well.

US 20030045495 is disclosing modified inducible systems for selectiveexpression of therapeutic genes by hyperthermia. However, hypothermia isalso difficult to be applied to discrete cells or small tissue areas.

US 20010049828 is describing a method and system for controlling theexpression of transgene products in specific tissues in a transgenicanimal. The system is based on an interaction of varioustransactivators. The transcactivator activity is controlled by antisensewhich is under control of tissue-specific promoters, thereby suppressingexpression in certain tissues. The system is rather complicated andrelies on serveral expression constructs and transgenic transcriptionfactors. A similar system is described in US 20020065243.

US 20020022018 described control of tissue-specificity by employingtissue-specific deletion or destruction of the expression-construct inthe target organism by tissue-specific expression of a Cre recombinase.As a result of Cre recombinase expression, the same or another vectorthat expresses the transgene in that tissue will be cut by the action ofthe Cre recombinase into several pieces due to LoxP sites that arestrategically placed within the vector backbone. Consequently, unwantedtransgene as well as viral gene expression are prevented. However, dueto leakiness of the promoter driving Cre expression, expression isexpected to be lowered also in the target tissue itself, therebydecreasing overall efficiency of this approach.

Although each of the afore-mentioned systems display inducibilitythereby solving problem with the temporal control of gene expression,the spatial precision of gene induction is still lacking. All systemsdisclosed in the art so far are either highly complex and/or alsoreducing efficient expression in the target cells. Thus, there remainssubstantial need for improvement of tissue-specificity or control ofpromoter leakiness. The present invention provides such means andmethods thereby fulfilling this longstanding need and desire in the art.

SUMMARY OF THE INVENTION

A first embodiment of the invention relates to a method for transgenicexpression with enhanced specificity in an eukaryotic organism saidmethod comprising the steps of:

-   a) providing an expression construct comprising a promoter sequence    functional in said eukaryotic organism and functionally linked    thereto a nucleotide sequence to be expressed into a chimeric RNA    sequence, said nucleotide sequence comprising    -   i) at least one sequence capable to confer a preferred phenotype        or beneficial effect to said eukaryotic organism, and    -   ii) at least one sequence substantially complementary to a        microRNA sequence naturally expressed in said eukaryotic        organism, wherein said microRNA is naturally expressed in        tissues, at times, and/or under environmental conditions, where        expression is not desired, but is not or substantially less        expressed in tissues, at times, and/or under environmental        conditions, where such expression is desired, wherein at least        one of sequence i) and sequence ii) are heterologous to each        other, and-   b) introducing said expression construct into an eukaryotic    organism.

Preferably, said eukaryotic organism is a human, an animal or a plant.

Various positions are possible for the sequence being substantiallycomplementary to the microRNA (hereinafter also the “microRNA tag”) inthe nucleotide sequence to be expressed. Preferably, the sequence beingsubstantially complementary to the microRNA is positioned in a locationof the nucleotide sequence to be expressed corresponding to the5′-untranslated region or the 3′-untranslated region of said sequence.

The nucleotide sequence to be expressed may have various form and/orfunctions. For example, it may comprise an open reading frame encoding aprotein. Alternatively, it may encode a functional RNA selected from thegroup consisting of antisense RNA, sense RNA, double-stranded RNA orribozymes. Said functional RNA is preferably attenuating expression ofan endogenous gene.

To allow for enhanced expression specificity, the microRNA (to which thesequence comprised in the nucleotide sequence to be expressed issubstantially complementary) is preferably not constitutively expressed,but is varying in expression in at least one parameter selected from thegroup consisting of tissue, special, time, development, environmental orother exogenous factors. Preferably, the microRNA is tissue-specificexpressed, spatially regulated, developmentally regulated, and/orregulated by biotic or abiotic stress factors.

The expression construct for the expression of the nucleotide sequencecomprising the microRNA-tag can be RNA, RNA and can be single- ordouble-stranded. Preferably the expression construct is DNA, morepreferably double-stranded DNA. The expression construct can be part ora larger vector construct. Preferably, the expression construct is in aplasmid.

Various promoters can be used for expression of the nucleotide sequencecomprising the microRNA-tag. The promoters can—for example—be selectedfrom the group consisting of constitutive promoters, tissue-specific ortissue-preferential promoters, and inducible promoters. Atissue-specific promoter in this context, does—preferably—mean which isleaky (i.e. having expression activity in other than the preferred ormain tissue) to a small but measurable extent.

The invention has broad opportunities of application, both in the fieldof plants, human and animals.

In one preferred embodiment, the eukaryotic organism is a plant and thepromoter is a promoter functional in plants. For plants, the expressednucleotide sequence preferably modulates expression of a gene involvedin agronomic traits, disease resistance, herbicide resistance, and/orgrain characteristics. The person skilled in art is aware of numerousnucleotide sequences which can be used in the context and for which aenhanced expression specificity is advantageous. For example, theexpressed nucleotide sequence may modulate expression of a gene selectedfrom the group consisting of genes involved in the synthesis and/ordegradation of proteins, peptides, fatty acids, lipids, waxes, oils,starches, sugars, carbohydrates, flavors, odors, toxins, carotenoids,hormones, polymers, flavinoids, storage proteins, phenolic acids,alkaloids, lignins, tannins, celluloses, glycoproteins, and glycolipids.

Various applications in plants are contemplated herein for whichmodulation of the expression profile in certain directions isadvantageous. This modulation is achieved by selection the microRNA-tagin a way, that the expression profile of the naturally occurring miRNAfits with the tissues, times, and/or under environmental conditionswhere no or lower expression should be achieved. For example, themicroRNA has a natural expression profile in the plant selected from thegroup consisting of

-   a) substantially constitutive expression but no expression in seed,-   b) predominant expression in seeds but not in other tissues,-   b) drought or other abiotic stress—induced expression,-   c) plant pathogen—induced expression,-   c) temporal expression (e.g., during early development, germination,    pollination etc.), and-   d) chemical induced expression.

Preferably, the microRNA is a plant microRNA selected from the groupconsisting of

-   a) the sequences as described by SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8,    9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,    26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,    43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 225, 226, 227,    228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240,    241, 242, 243, 245, 245, 246, 247, 248, 249, 250, 251, 252, 253,    254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, and 266    and-   b) derivatives of the sequences described by SEQ ID NO: 1, 2, 3, 4,    5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,    23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,    40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 225,    226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238,    239, 240, 241, 242, 243, 245, 245, 246, 247, 248, 249, 250, 251,    252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264,    265, and 266.

In one preferred embodiment, said derivate has an identity of at least70%, preferably at least 80% or 85%, more preferably at least 90%, mostpreferably at least 95% to a sequence described by any of SEQ ID NO: 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 225,226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239,240, 241, 242, 243, 245, 245, 246, 247, 248, 249, 250, 251, 252, 253,254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, and 266.

Other applications of the invention provide herein are used in animals(especially mammals) or human. Especially preferred are pharmaceuticalapplications. Thus, in another preferred embodiment of the invention thetarget organism is a mammal (more preferably a human being) and thepromoter is a promoter functional in mammals (more preferably inhumans). The expressed nucleotide sequence comprising the miRNA-tagpreferably modulates (e.g., express, over-express, or suppress)expression of a gene selected from the group consisting of genesinvolved in a human or animal disease or is a therapeutic gene.Alternatively, exogenous genes or sequences may be expressed which havea curative effect on the target organism. The disease is preferablyselected from the group of immunological diseases, cancer, diabetes,neurodegeneration, and metabolism diseases. The person skilled in theart is aware of numerous sequences, which can be used in this context.The modulated gene may be selected from the group consisting ofretinoblastoma protein, p53, angiostatin, leptin, hormones, growthfactors, cytokines, insulin, growth hormones, alpha-interferon,beta-glucocerebrosidase, serum albumin, hemoglobin, and collagen.Therapeutic genes may be selected from the group consisting of tumornecrosis factor alpha. In this context the invention disclosed herein isa improved method for gene therapy or nucleotide-mediated therapy.

Various promoters are currently used in the art to express sequences inanimal, mammalian or human organism. Most of them are lackingtissue-specificity and can be advantageously combined with the teachingprovided herein. For example the promoter may be selected from groupconsisting of the perbB2 promoter, whey acidic protein promoter,stromelysin 3 promoter, prostate specific antigen promoter, probasinpromoter.

Various applications in animal, mammalian or human organisms arecontemplated herein for which modulation of the expression profile incertain directions is advantageous. This modulation is achieved byselection the microRNA-tag in a way, that the expression profile of thenaturally occurring miRNA fits with the tissues, times, and/or underenvironmental conditions where no or lower expression should beachieved. For example, the microRNA has a natural expression profile inthe animal, mammalian or human organism selected from the groupconsisting of

-   a) tissue-specific expression in a tissue selected from the group    consisting of brain tissue, liver tissue, muscle tissue, neuron    tissue, and tumor tissue.-   b) stress-induced expression,-   c) pathogen-induced expression,-   d) neoplastic growth or tumorgenic growth induced expression, and-   e) age-dependent expression.

Preferably, the microRNA is an animal, mammalian or human microRNAselected from the group consisting of

-   a) the sequences as described by SEQ ID NO: 56, 57, 58, 59, 60, 61,    62, and 63, and-   b) derivatives of the sequences described by SEQ ID NO: 56, 57, 58,    59, 60, 61, 62, and 63, and-   c) the complementary RNA sequence to a sequence as described by any    of SEQ ID NO: 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102,    103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,    116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,    129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,    142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,    155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167,    168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180,    181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193,    194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206,    207, or 208, and-   d) derivatives of RNA sequence complementary to a sequence as    described by any of SEQ ID NO: 90, 91, 92, 93, 94, 95, 96, 97, 98,    99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,    113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,    126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138,    139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151,    152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164,    165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177,    178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190,    191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203,    204, 205, 206, 207, or 208.

In one preferred embodiment, said derivate has an identity of at least70%, preferably at least 80% or 85%, more preferably at least 90%, mostpreferably at least 95% to a miRNA as described by any of SEQ ID NO: 56,57, 58, 59, 60, 61, 62, and 63 or a RNA sequence complementary to asequence as described by any of SEQ ID NO: 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139,140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153,154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167,168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181,182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195,196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, or 208.

The chimeric RNA expressed by the method of the invention (i.e. the RNAcomprising the expressed miRNA-tag) is considered to be novel. Thusanther embodiment of the invention relates to a chimeric ribonucleotidesequence comprising

-   i) at least one sequence capable to confer a preferred phenotype or    beneficial effect to a eukaryotic organism, and-   ii) at least one sequence substantially complementary to a microRNA    sequence naturally occurring in a eukaryotic organism,    wherein at least one of sequence i) and sequence ii) are    heterologous to each other.

The sequences i) and/or ii) in said chimeric ribonucleotide sequence arepreferably defined as above for the method of the invention.

Furthermore, the expression constructs for expression of said chimericribonucleotide sequence (which are employed in the method of theinvention) are considered to be novel. Thus another embodiment of theinvention relates to an expression construct comprising a promotersequence functional in a eukaryotic organism and functionally linkedthereto a nucleotide sequence to be expressed, said sequence comprising

-   i) at least one sequence capable to confer a preferred phenotype or    beneficial effect to said eukaryotic organism, and-   ii) at least one sequence substantially complementary to a microRNA    sequence naturally occurring in said eukaryotic organism,    wherein at least one of sequence i) and sequence ii) are    heterologous to each other.

The expression construct and its elements are preferably defined asabove for the method of the invention.

Another embodiment of the invention relates to an expression vectorcomprising an expression construct of the invention. Preferably, theexpression vector is an eukaryotic expression vector. More preferablythe eukaryotic expression vector is a viral vector, a plasmid vector ora binary vector.

Yet another embodiment of the invention relates to a transformed cell ororganism (preferably a non-human organism) comprising a chimericribonucleotide sequence, an expression construct or an expression vectorof the invention. Preferably, said expression construct or expressionvector are inserted (at least in part) into the genome of the cell ororganism. Preferably, said cell or organism is selected from the groupof mammalian, bacterial, fungal, nematode or plant cells and organism.Another embodiment of the invention relates to transformed seed of theplant of the invention.

Yet another embodiment of the invention relates to a pharmaceuticallypreparation of at least one expression construct, a chimericribonucelotide sequence, or a vector according to the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the matter in which the above-recited features, advantages andobjects of the invention, as well as others which will become clear, areattained and can be understood in detail, more particular descriptionsof the invention briefly summarized above may be had by reference tocertain embodiments thereof which are illustrated in the appendeddrawings. These drawings form a part of the specification. It is to benoted, however, that the appended drawings illustrate preferredembodiments of the invention and therefore are not to be consideredlimiting in their scope.

FIG. 1 Biogenesis and Mode of Action of miRNAs in Plant (Bartel D, Cell116:281-297, 2004)

-   -   Step 1: A miRNA gene is transcribed into Pri-miRNA by Pol II.        There is an increasing evidence that, at least, some transcripts        have Cap structure at 5′ terminus and are polyadenylylated.at 3′        terminus. The short-lived Pri-miRNA forms a stem-loop structure        and quickly enters into Step 2.    -   Step 2: Pri-miRNA is processed into Pre-miRNA by Dicer-1        resulting in exposure of one end of mature miRNA.    -   Step 3: Pre-miRNA is processed into mature miRNA:miRNA* duplex        (approx. 22 nt) by DCL1 or another gene.    -   Step 4: miRNA is exported from nucleus into cytoplasm. Likely,        HASTY, the plant orthologue of mammalian Exportin-5, is required        for such exporting process.    -   Step 5,6: A single-stranded miRNA is eventually incorporated        into RISC (RNA-induced silencing complex) and binds specifically        to target mRNA with perfect or near perfect sites complimentary        to miRNA.    -   Step 7: miRNA inhibits gene expression at post-transcription        levels or translational levels.

FIG. 2 Specific Expression Patterns of Maize microRNA precursors in Zeamays gene expression database.

-   -   A: Expression of miR166 Precursor in Leaves and Tassel    -   B: Predominate Expression of miR167 Precursor in Seeds    -   C: Expression of miR159 Precursor in Everywhere but Seeds    -   D: Stress Induced Expression of miR160 Precursor

FIG. 3 Enhancing seed-specific expression

Maize miR159 is expressed in all tissues except (FIG. 3-A). If the geneof interest (GOI) is intended to express only in seeds with leaky‘seed-specific’ promoter, one can incorporated a miRNA-tag(5′-AGAGCTCCCTTCAATCCAAA-3′ (SEQ ID NO: 267), which is complementary tomiR159 (SEQ ID NO: 268)) into 3′UTR following the GOI to make a genericbinary vector to control leaky expression of the GOI in non-seed tissuesby endogenous miR159 (FIG. 3-B). The GOI is only efficiently expressedin seeds, but its mRNA is broken down (symbolized by the pairs ofscissors) in other tissues, where the endogenous miRNA159 is expressed.

FIG. 4 Enhancing specificity of expression in non-seed tissues(preventing expression seeds)

Maize miR167 is predominantly expressed in seeds (FIG. 4-a). If the geneof interest (GOI) is NOT intended to express in seeds (e.g., genesconferring pesticide activities), but promoter used is leaky in seeds,one can incorporate a tag (5′-TGAAGCTGCCAGCATGATCT-3′ (SEQ ID NO: 269),complementary to miR167 (SEQ ID NO: 270)) into the 3′ UTR following theGOI to make a generic vector to control undesirable expression of theGOI in seeds by endogenous miR167 (FIG. 4-B). The GOI is onlyefficiently broken down in seeds (symbolized by the pair of scissors),where the endogenous miRNA167 is expressed, in other tissues the GOI isexpressed.

FIG. 5 A Generic Vector to Control Leakiness of GOI Expression

-   -   The invention disclosed herein can be employed to regulate        transgene expression in spatial and/or temporal manner. Some        traits (e.g. for animal feed) require the gene of interest (GOI)        to express in certain stages (e.g. early or late embryos).        Certain miRNAs could be regulated at different developmental        stages. Therefore, one can incorporate miRNA target sites that        are complementary to miRNA X (tissue-specific) and/or miRNA Y        (developmental specific), so that expression of GOI can be        controlled as a most desirable way.

DEFINITIONS

Abbreviations: BAP—6-benzylaminopurine; 2,4-D-2,4-dichlorophenoxyaceticacid; MS—Mura-shige and Skoog medium; NAA—1-naphtaleneacetic acid; MES,2-(N-morpholinoethanesulfonic acid, IAA indole acetic acid; Kan:Kanamycin sulfate; GA3—Gibberellic acid; Timentin™: ticarcillindisodium/clavulanate potassium.

It is to be understood that this invention is not limited to theparticular methodology, protocols, cell lines, plant species or genera,constructs, and reagents described as such. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention which will be limited only by the appended claims.It must be noted that as used herein and in the appended claims, thesingular forms “a,” “and,” and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to “avector” is a reference to one or more vectors and includes equivalentsthereof known to those skilled in the art, and so forth. The term“about” is used herein to mean approximately, roughly, around, or in theregion of. When the term “about” is used in conjunction with a numericalrange, it modifies that range by extending the boundaries above andbelow the numerical values set forth. In general, the term “about” isused herein to modify a numerical value above and below the stated valueby a variance of 20 percent, preferably 10 percent up or down (higher orlower). As used herein, the word “or” means any one member of aparticular list and also includes any combination of members of thatlist. The words “comprise,” “comprising,” “include,” “including,” and“includes” when used in this specification and in the following claimsare intended to specify the presence of one or more stated features,integers, components, or steps, but they do not preclude the presence oraddition of one or more other features, integers, components, steps, orgroups thereof. For clarity, certain terms used in the specification aredefined and used as follows:

Agronomically valuable trait: The term “agronomically valuable trait”refers to any phenotype in a plant organism that is useful oradvantageous for food production or food products, including plant partsand plant products. Non-food agricultural products such as paper, etc.are also included. A partial list of agronomically valuable traitsincludes pest resistance, vigor, development time (time to harvest),enhanced nutrient content, novel growth patterns, flavors or colors,salt, heat, drought and cold tolerance, and the like. Preferably,agronomically valuable traits do not include selectable marker genes(e.g., genes encoding herbicide or antibiotic resistance used only tofacilitate detection or selection of transformed cells), hormonebiosynthesis genes leading to the production of a plant hormone (e.g.,auxins, gibberllins, cytokinins, abscisic acid and ethylene that areused only for selection), or reporter genes (e.g. luciferase,glucuronidase, chloramphenicol acetyl transferase (CAT, etc.). Suchagronomically valuable important traits may indude improvement of pestresistance (e.g., Melchers et al. (2000) Curr Opin Plant Biol3(2):147-52), vigor, development time (time to harvest), enhancednutrient content, novel growth patterns, flavors or colors, salt, heat,drought, and cold tolerance (e.g., Sakamoto et al. (2000) J Exp Bot51(342):81-8; Saijo et al. (2000) Plant J 23(3): 319-327; Yeo et al.(2000) Mol Cells 10(3):263-8; Cushman et al. (2000) Curr Opin Plant Biol3(2):117-24), and the like. Those of skill will recognize that there arenumerous polynucleotides from which to choose to confer these and otheragronomically valuable traits.

Alter: To “alter” or “modulate” the expression of a nucleotide sequencein a cell (e.g., a plant cell) means that the level of expression of thenucleotide sequence in a cell after applying a method of the presentinvention is different from its expression in the cell before applyingthe method. In a preferred embodiment, to alter expression means thatthe expression of the nucleotide sequence in the plant is reduced afterapplying a method of the present invention as compared to beforeapplying the method. “Reduction of” or “to reduce” the expression of atarget gene is to be understood in the broad sense and comprises thepartial or essentially complete prevention or blocking of the expressionof the target gene or the RNA, mRNA, rRNA, tRNA derived therefrom and/orof the protein product encoded by it in a cell, an organism or a part,tissue, organ, cell or seed thereof, which prevention or blockage may bebased on different cell-biological mechanisms. The term “reduced” meansherein lower, preferably significantly lower, more preferably theexpression of the nucleotide sequence is not detectable. As used herein,“a reduction” of the level of an agent such as a protein or mRNA meansthat the level is reduced relative to a cell or organism lacking achimeric RNA molecule of the invention capable of reducing the agent. Asused herein, “at least a partial reduction” of the level of an agent(such as a RNA, mRNA, rRNA, tRNA expressed by the target gene and/or ofthe protein product encoded by it) means that the level is reduced atleast 25%, preferably at least 50%, relative to a cell or organismlacking a chimeric RNA molecule of the invention capable of reducingsaid agent. As used herein, “a substantial reduction” of the level of anagent such as a protein or mRNA means that the level is reduced relativeto a cell or organism lacking a chimeric RNA molecule of the inventioncapable of reducing the agent, where the reduction of the level of theagent is at least 75%, preferably at least 85%. As used herein, “aneffective elimination” of an agent such as a protein or mRNA is relativeto a cell or organism lacking a chimeric RNA molecule of the inventioncapable of reducing the agent, where the reduction of the level of theagent is greater than 95%, preferably greater than 98%. The reductioncan be determined by methods with which the skilled worker is familiar.Thus, the reduction of the protein quantity can be determined forexample by an immunological detection of the protein. Moreover,biochemical techniques such as Northern hybridization, nucleaseprotection assay, reverse transcription (quantitative RT-PCR), ELISA(enzyme-linked immunosorbent assay), Western blotting, radioimmunoassay(RIA) or other immunoassays and fluorescence-activated cell analysis(FACS) can be employed. Depending on the type of the reduced proteinproduct, its activity or the effect on the phenotype of the organism orthe cell may also be determined. Methods for determining the proteinquantity are known to the skilled worker. Examples, which may bementioned, are: the micro-Biuret method (Goa J (1953) Scand J Clin LabInvest 5:218-222), the Folin-Ciocalteau method (Lowry O H et al. (1951)J Biol Chem 193:265-275) or measuring the absorption of CBB G-250(Bradford M M (1976) Analyt Biochem 72:248-254). In another preferredembodiment, to alter expression means that the expression of thenucleotide sequence in the plant is increased after applying a method ofthe present invention as compared to before applying the method.

Amino acid sequence: As used herein, the term “amino acid sequence”refers to a list of abbreviations, letters, characters or wordsrepresenting amino acid residues. Amino acids may be referred to hereinby either their commonly known three letter symbols or by the one-lettersymbols recommended by the IUPAC-IUB Biochemical NomenclatureCommission. Nucleotides, likewise, may be referred to by their commonlyaccepted single-letter codes.

Animal: The terms “animal” or “animal organism” refer to nonhumanvertebrates or invertebrates. Preferred vertebrates comprise, forexample, fish species, nonhuman mammals such as cattle, horse, sheep,goat, mouse, rat or pig, and birds such as chicken or goose. Preferredanimal cells comprise CHO, COS, HEK293 cells. Invertebrates comprisenematodes or other worms, and insects. Invertebrates comprise insectcells such as Drosophila S2 and Spodoptera Sf9 or Sf21 cells.Furthermore preferred are nematodes, which are capable of attackinganimals or humans, such as those of the genera Ancylostoma, Ascaridia,Ascaris, Bunostomum, Caenorhabditis, Capillaria, Chabertia, Cooperia,Dictyocaulus, Haemonchus, Heterakis, Nematodirus, Oesophagostomum,Ostertagia, Oxyuris, Parascaris, Strongylus, Toxascaris, Trichuris,Trichostrongylus, Tfhchonema, Toxocara or Uncinaria. Furthermorepreferred are those which are capable of attacking plant organisms suchas, for example, Bursaphalenchus, Criconemella, Diiylenchus,Ditylenchus, Globodera, Helicotylenchus, Heterodera, Longidorus,Melodoigyne, Nacobbus, Paratylenchus, Pratylenchus, Radopholus,Rotelynchus, Tylenchus or Xiphinema. Preferred insects comprise those ofthe genera Coleoptera, Diptera, Lepidoptera and Homoptera.

Antiparallel: “Antiparallel” refers herein to two nucleotide sequencespaired through hydrogen bonds between complementary base residues withphosphodiester bonds running in the 5′-3′ direction in one nucleotidesequence and in the 3′-5′ direction in the other nucleotide sequence.

Antisense: The term “antisense” refers to a nucleotide sequence that isinverted relative to its normal orientation for transcription and soexpresses an RNA transcript that is complementary to a target gene mRNAmolecule expressed within the host cell (e.g., it can hybridize to thetarget gene mRNA molecule through Watson-Crick base pairing). Anantisense strand may be constructed in a number of different ways,provided that it is capable of interfering with the expression of atarget gene. For example, the antisense strand can be constructed byinverting the coding region (or a portion thereof) of the target generelative to its normal orientation for transcription to allow thetranscription of its complement, (e.g., RNAs encoded by the antisenseand sense gene may be complementary). Furthermore, the antisenseoligonucleotide strand need not have the same intron or exon pattern asthe target gene, and noncoding segments of the target gene may beequally effective in achieving antisense suppression of target geneexpression as coding segments. In the context of gene silencing the term“antisense” is understood to mean a nucleic acid having a sequencecomplementary to a target sequence, for example a messenger RNA (mRNA)sequence the blocking of whose expression is sought to be initiated byhybridization with the target sequence.

Cell: The term “cell” or “plant cell” as used herein refers preferablyto a single cell. The term “cells” refers to a population of cells. Thepopulation may be a pure population comprising one cell type. Likewise,the population may comprise more than one cell type. In the presentinvention, there is no limit on the number of cell types that a cellpopulation may comprise. The cells may be synchronized or notsynchronized. A cell within the meaning of this invention may beisolated (e.g., in suspension culture) or comprised in a tissue, organor organism at any developmental stage.

Coding region: As used herein the term “coding region” when used inreference to a structural gene refers to the nucleotide sequences whichencode the amino acids found in the nascent polypeptide as a result oftranslation of a mRNA molecule. The coding region is bounded, ineukaryotes, on the 5′-side by the nucleotide triplet “ATG” which encodesthe initiator methionine and on the 3′-side by one of the three tripletswhich specify stop codons (i.e., TAA, TAG, TGA). In addition tocontaining introns, genomic forms of a gene may also include sequenceslocated on both the 5′- and 3′-end of the sequences which are present onthe RNA transcript. These sequences are referred to as “flanking”sequences or regions (these flanking sequences are located 5′ or 3′ tothe non-translated sequences present on the mRNA transcript). The5′-flanking region may contain regulatory sequences such as promotersand enhancers which control or influence the transcription of the gene.The 3′-flanking region may contain sequences which direct thetermination of transcription, post-transcriptional cleavage andpolyadenylation.

Complementary: “Complementary” or “complementarity” refers to twonucleotide sequences which comprise antiparallel nucleotide sequencescapable of pairing with one another (by the base-pairing rules) uponformation of hydrogen bonds between the complementary base residues inthe antiparallel nucleotide sequences. For example, the sequence5′-AGT-3′ is complementary to the sequence 5′-ACT-3′. Complementaritycan be “partial” or “total.” “Partial” complementarity is where one ormore nucleic acid bases is not matched according to the base pairingrules. “Total” or “complete” complementarity between nucleic acids iswhere each and every nucleic acid base is matched with another baseunder the base pairing rules. The degree of complementarity betweennucleic acid strands has significant effects on the efficiency andstrength of hybridization between nucleic acid strands. A “complement”of a nucleic acid sequence as used herein refers to a nucleotidesequence whose nucleic acids show total complementarity to the nucleicacids of the nucleic acid sequence.

Chromosomal DNA: The term “chromosomal DNA” or “chromosomalDNA-sequence” is to be understood as the genomic DNA of the cellularnucleus independent from the cell cycle status. Chromosomal DNA mighttherefore be organized in chromosomes or chromatids, they might becondensed or uncoiled. An insertion into the chromosomal DNA can bedemonstrated and analyzed by various methods known in the art like e.g.,polymerase chain reaction (PCR) analysis, Southern blot analysis,fluorescence in situ hybridization (FISH), and in situ PCR.

DNA shuffling: DNA shuffling is a method to rapidly, easily andefficiently introduce mutations or rearrangements, preferably randomly,in a DNA molecule or to generate exchanges of DNA sequences between twoor more DNA molecules, preferably randomly. The DNA molecule resultingfrom DNA shuffling is a shuffled DNA molecule that is a non-naturallyoccurring DNA molecule derived from at least one template DNA molecule.The shuffled DNA encodes an enzyme modified with respect to the enzymeencoded by the template DNA, and preferably has an altered biologicalactivity with respect to the enzyme encoded by the template DNA.

Double-stranded RNA: A “double-stranded RNA” molecule, “RNAi molecule”,or “dsRNA” molecule comprises a sense RNA fragment of a nucleotidesequence and an antisense RNA fragment of the nucleotide sequence, whichboth comprise nucleotide sequences complementary to one another, therebyallowing the sense and antisense RNA fragments to pair and form adouble-stranded RNA molecule. Preferably the terms refer to adouble-stranded RNA molecule capable, when introduced into a cell ororganism, of at least partially reducing the level of an mRNA speciespresent in a cell or a cell of an organism. As used herein, “RNAinterference”, “RNAi, and “dsRNAi” refer to gene-specific silencing thatis induced by the introduction of a double-stranded RNA molecule.

Endogenous: An “endogenous” nucleotide sequence refers to a nucleotidesequence, which is present in the genome of the untransformed cell(e.g., a plant or mammalian cell).

Essential: An “essential” gene is a gene encoding a protein such as e.g.a biosynthetic enzyme, receptor, signal transduction protein, structuralgene product, or transport protein that is essential to the growth orsurvival of the organism or cell (e.g., a plant).

Exon: The term “exon” as used herein refers to the normal sense of theterm as meaning a segment of nucleic acid molecules, usually DNA, thatencodes part of or all of an expressed protein.

Expression: “Expression” refers to the biosynthesis of a gene product,preferably to the transcription and/or translation of a nucleotidesequence, for example an endogenous gene or a heterologous gene, in acell. For example, in the case of a structural gene, expression involvestranscription of the structural gene into mRNA and—optionally—thesubsequent translation of mRNA into one or more polypeptides. In thecase of antisense constructs, for example, expression may refer to thetranscription of the antisense DNA only.

Expression construct/expression construct: “Expression construct” and“expression construct” as used herein are synonyms and mean a DNAsequence capable of directing expression of a particular nucleotidesequence in an appropriate host cell (e.g., a plant pr mammalian cell),comprising a promoter functional in said host cell into which it will beintroduced, operatively linked to the nucleotide sequence of interestwhich is—optionally—operatively linked to termination signals. Iftranslation is required, it also typically comprises sequences requiredfor proper translation of the nucleotide sequence. The coding region maycode for a protein of interest but may also code for a functional RNA ofinterest, for example antisense RNA, dsRNA, or a nontranslated RNA, inthe sense or antisense direction. The expression construct comprisingthe nucleotide sequence of interest may be chimeric, meaning that atleast one of its components is heterologous with respect to at least oneof its other components. The expression construct may also be one, whichis naturally occurring but has been obtained in a recombinant formuseful for heterologous expression. Typically, however, the expressionconstruct is heterologous with respect to the host, i.e., the particularDNA sequence of the expression construct does not occur naturally in thehost cell and must have been introduced into the host cell or anancestor of the host cell by a transformation event. The expression ofthe nucleotide sequence in the expression construct may be under thecontrol of a constitutive promoter or of an inducible promoter, whichinitiates transcription only when the host cell is exposed to someparticular external stimulus. In the case of a multicellular organism,such as a plant, the promoter can also be specific to a particulartissue or organ or stage of development.

Foreign gene: The term “foreign gene” refers to any nucleic acid (e.g.,gene sequence) which is introduced into the genome of a cell byexperimental manipulations and may include gene sequences found in thatcell so long as the introduced gene contains some modification (e.g., apoint mutation, the presence of a selectable marker gene, etc.) relativeto the naturally-occurring gene.

Gene: The term “gene” refers to a coding region operably joined toappropriate regulatory sequences capable of regulating the expression ofthe gene product (e.g., a polypeptide or a functional RNA) in somemanner. A gene includes untranslated regulatory regions of DNA (e.g.,promoters, enhancers, repressors, etc.) preceding (up-stream) andfollowing (downstream) the coding region (open reading frame, ORF) aswell as, where applicable, intervening sequences (i.e., introns) betweenindividual coding regions (i.e., exons). The term “structural gene” asused herein is intended to mean a DNA sequence that is transcribed intomRNA which is then translated into a sequence of amino acidscharacteristic of a specific polypeptide.

Genetically modified organism: The term “genetically-modified organism”or “GMO” refers to any organism that comprises heterologous DNA or atransgene. Exemplary organisms include plants, animals andmicroorganisms.

Genome and genomic DNA: The terms “genome” or “genomic DNA” is referringto the heritable genetic information of a host organism. Said genomicDNA comprises the DNA of the nucleus (also referred to as chromosomalDNA) but also the DNA of the plastids (e.g., chloroplasts) and othercellular organelles (e.g., mitochondria). Preferably the terms genome orgenomic DNA is referring to the chromosomal DNA of the nucleus.

Hairpin RNA: As used herein “hairpin RNA” refers to any self-annealingdouble stranded RNA molecule. In its simplest representation, a hairpinRNA consists of a double stranded stem made up by the annealing RNAstrands, connected by a single stranded RNA loop, and is also referredto as a “pan-handle RNA”. However, the term “hairpin RNA” is alsointended to encompass more complicated secondary RNA structurescomprising self-annealing double stranded RNA sequences, but alsointernal bulges and loops. The specific secondary structure adapted willbe determined by the free energy of the RNA molecule, and can bepredicted for different situations using appropriate software such asFOLDRNA (Zuker and Stiegler (1981) Nucleic Acids Res 9(1):133-48; Zuker,M. (1989) Methods Enzymol. 180, 262-288).

Heterologous: The terms “heterologous” with respect to a nucleic acid orDNA refer to a nucleotide sequence which is ligated to, or ismanipulated to become ligated to, a nucleic acid sequence to which it isnot ligated in nature, or to which it is ligated at a different locationin nature. A heterologous expression construct comprising a nucleic acidsequence and at least one regulatory sequence (such as an promoter or antranscription termination signal) linked thereto for example is aconstructs originating by experimental manipulations in which either a)said nucleic acid sequence, or b) said regulatory sequence or c) both(i.e. (a) and (b)) is not located in its natural (native) geneticenvironment or has been modified by experimental manipulations, anexample of a modification being a substitution, addition, deletion,inversion or insertion of one or more nucleotide residues. Naturalgenetic environment refers to the natural chromosomal locus in theorganism of origin, or to the presence in a genomic library. In the caseof a genomic library, the natural genetic environment of the nucleicacid sequence is preferably retained, at least in part. The environmentflanks the nucleic acid sequence at least at one side and has a sequenceof at least 50 bp, preferably at least 500 bp, especially preferably atleast 1,000 bp, very especially preferably at least 5,000 bp, in length.A naturally occurring expression construct—for example the naturallyoccurring combination of a promoter with the corresponding gene—becomesa transgenic expression construct when it is modified by non-natural,synthetic “artificial” methods such as, for example, mutagenization.Such methods have been described (U.S. Pat. No. 5,565,350; WO 00/15815).For example a protein encoding nucleic acid sequence operably lined to apromoter, which is not the native promoter of this sequence, isconsidered to be heterologous with respect to the promoter. Preferably,heterologous DNA is not endogenous to or not naturally associated withthe cell into which it is introduced, but has been obtained from anothercell. Heterologous DNA also includes an endogenous DNA sequence, whichcontains some modification, non-naturally occurring multiple copies of aendogenous DNA sequence, or a DNA sequence which is not naturallyassociated with another DNA sequence physically linked thereto.Generally, although not necessarily, heterologous DNA encodes RNA andproteins that are not normally produced by the cell into which it isexpressed.

Homologous DNA Sequence: a DNA sequence naturally associated with a hostcell or another DNA sequence.

Hybridization: The term “hybridization” as used herein includes “anyprocess by which a strand of nucleic acid joins with a complementarystrand through base pairing.” (J. Coombs (1994) Dictionary ofBiotechnology, Stockton Press, New York). Hybridization and the strengthof hybridization (i.e., the strength of the association between thenucleic acids) is impacted by such factors as the degree ofcomplementarity between the nucleic acids, stringency of the conditionsinvolved, the Tm of the formed hybrid, and the G:C ratio within thenucleic acids. As used herein, the term “Tm” is used in reference to the“melting temperature.” The melting temperature is the temperature atwhich a population of double-stranded nucleic acid molecules becomeshalf dissociated into single strands. The equation for calculating theTm of nucleic acids is well known in the art. As indicated by standardreferences, a simple estimate of the Tm value may be calculated by theequation: Tm=81.5+0.41(% G+C), when a nucleic acid is in aqueoussolution at 1 M NaCl [see e.g., Anderson and Young, Quantitative FilterHybridization, in Nucleic Acid Hybridization (1985)]. Other referencesinclude more sophisticated computations, which take structural as wellas sequence characteristics into account for the calculation of Tm.Stringent conditions, are known to those skilled in the art and can befound in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.(1989), 6.3.1-6.3.6. Low stringency conditions when used in reference tonucleic acid hybridization comprise conditions equivalent to binding orhybridization at 68° C. in a solution consisting of 5×SSPE (43.8 g/LNaCl, 6.9 g/L NaH₂PO₄.H₂O and 1.85 g/L EDTA, pH adjusted to 7.4 withNaOH), 1% SDS, 5×Denhardt's reagent [50×Denhardt's contains thefollowing per 500 mL 5 g Ficoll (Type 400, Pharmacia), 5 g BSA (FractionV; Sigma)] and 100 μg/mL denatured salmon sperm DNA followed by washing(preferably for one times 15 minutes, more preferably two times 15minutes, more preferably three time 15 minutes) in a solution comprising1×SSC (1×SSC is 0.15 M NaCl plus 0.015 M sodium citrate) and 0.1% SDS atroom temperature or—preferably 37° C.—when a DNA probe of preferablyabout 100 to about 1,000 nucleotides in length is employed. Mediumstringency conditions when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 68° C. in a solution consisting of 5×SSPE (43.8 g/L NaCl, 6.9 g/LNaH₂PO₄.H₂O and 1.85 g/L EDTA, pH adjusted to 7.4 with NaOH), 1% SDS,5×Denhardt's reagent [50×Denhardt's contains the following per 500 mL 5g Ficoll (Type 400, Pharmacia), 5 g BSA (Fraction V; Sigma)] and 100μg/mL denatured salmon sperm DNA followed by washing (preferably for onetimes 15 minutes, more preferably two times 15 minutes, more preferablythree time 15 minutes) in a solution comprising 0.1×SSC (1×SSC is 0.15 MNaCl plus 0.015 M sodium citrate) and 1% SDS at room temperatureor—preferably 37° C.—when a DNA probe of preferably about 100 to about1,000 nucleotides in length is employed. High stringency conditions whenused in reference to nucleic acid hybridization comprise conditionsequivalent to binding or hybridization at 68° C. in a solutionconsisting of 5×SSPE, 1% SDS, 5×Denhardt's reagent and 100 μg/mLdenatured salmon sperm DNA followed by washing (preferably for one times15 minutes, more preferably two times 15 minutes, more preferably threetime 15 minutes) in a solution comprising 0.1×SSC, and 1% SDS at 68° C.,when a probe of preferably about 100 to about 1,000 nucleotides inlength is employed. The term “equivalent” when made in reference to ahybridization condition as it relates to a hybridization condition ofinterest means that the hybridization condition and the hybridizationcondition of interest result in hybridization of nucleic acid sequenceswhich have the same range of percent (%) homology. For example, if ahybridization condition of interest results in hybridization of a firstnucleic acid sequence with other nucleic acid sequences that have from80% to 90% homology to the first nucleic acid sequence, then anotherhybridization condition is said to be equivalent to the hybridizationcondition of interest if this other hybridization condition also resultsin hybridization of the first nucleic acid sequence with the othernucleic acid sequences that have from 80% to 90% homology to the firstnucleic acid sequence. When used in reference to nucleic acidhybridization the art knows well that numerous equivalent conditions maybe employed to comprise either low or high stringency conditions;factors such as the length and nature (DNA, RNA, base composition) ofthe probe and nature of the target (DNA, RNA, base composition, presentin solution or immobilized, etc.) and the concentration of the salts andother components (e.g., the presence or absence of formamide, dextransulfate, polyethylene glycol) are considered and the hybridizationsolution may be varied to generate conditions of either low or highstringency hybridization different from, but equivalent to, theabove-listed conditions. Those skilled in the art know that whereashigher stringencies may be preferred to reduce or eliminate non-specificbinding, lower stringencies may be preferred to detect a larger numberof nucleic acid sequences having different homologies.

“Identity”: The term “identity” is a relationship between two or morepolypeptide sequences or two or more nucleic acid molecule sequences, asdetermined by comparing the sequences. In the art, “identity” also meansthe degree of sequence relatedness between polypeptide or nucleic acidmolecule sequences, as determined by the match between strings of suchsequences. “Identity” as used herein can be measured between nucleicacid sequences of the same ribonucleic-type (such as between DNA and DNAsequences) or between different types (such as between RNA and DNAsequences). It should be understood that in comparing an RNA sequence toa DNA sequence, an “identical” RNA sequence will contain ribonucleotideswhere the DNA sequence contains deoxyribonucleotides, and further thatthe RNA sequence will contain a uracil at positions where the DNAsequence contains thymidine. In case an identity is measured between RNAand DNA sequences, uracil bases of RNA sequences are considered to beidentical to thymidine bases of DNA sequences. “Identity” can be readilycalculated by known methods including, but not limited to, thosedescribed in Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York (1988); Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M. and Griffin, H. G.,eds., Humana Press, New Jersey (1994); Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press (1987); Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., Stockton Press, New York(1991); and Carillo, H., and Lipman, D., SIAM J. Applied Math, 48:1073(1988). Methods to determine identity are designed to give the largestmatch between the sequences tested. Moreover, methods to determineidentity are codified in publicly available programs. Computer programswhich can be used to determine identity between two sequences include,but are not limited to, GCG (Devereux, J., et al., Nucleic AcidsResearch 12(1):387 (1984); suite of five BLAST programs, three designedfor nucleotide sequences queries (BLASTN, BLASTX, and TBLASTX) and twodesigned for protein sequence queries (BLASTP and TBLASTN) (Coulson,Trends in Biotechnology, 12:76-80 (1994); Birren et al., GenomeAnalysis, 1:543-559 (1997)). The BLASTX program is publicly availablefrom NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBINLM NIH, Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol.,215:403-410 (1990)). The well-known Smith Waterman algorithm can also beused to determine identity. Parameters for polypeptide sequencecomparison typically include the following:

-   -   Algorithm: Needleman and Wunsch, J. Mol. Biol., 48:443-453        (1970)    -   Comparison matrix: BLOSUM62 from Hentikoff and Hentikoff, Proc.        Natl. Acad. Sci. USA, 89:10915-10919 (1992)    -   Gap Penalty: 12    -   Gap Length Penalty: 4

A program, which can be used with these parameters, is publiclyavailable as the “gap” program from Genetics Computer Group, Madison,Wis. The above parameters along with no penalty for end gap are thedefault parameters for peptide comparisons. Parameters for nucleic acidmolecule sequence comparison include the following:

-   -   Algorithm: Needleman and Wunsch, J. Mol. Bio. 48:443-453 (1970)    -   Comparison matrix: matches-+10; mismatches=0    -   Gap Penalty: 50    -   Gap Length Penalty: 3

As used herein, “% identity” is determined using the above parameters asthe default parameters for nucleic acid molecule sequence comparisonsand the “gap” program from GCG, version 10.2.

Infecting: The terms “infecting” and “infection” with a bacterium orvirus refer to co-incubation of a target biological sample, (e.g., cell,tissue, etc.) with the bacterium or virus under conditions such thatnucleic acid sequences contained within the bacterium or virus areintroduced into one or more cells of the target biological sample.

Intron: The term “intron” as used herein refers to the normal sense ofthe term as meaning a segment of nucleic acid molecules, usually DNA,that does not encode part of or all of an expressed protein, and which,in endogenous conditions, is transcribed into RNA molecules, but whichis spliced out of the endogenous RNA before the RNA is translated into aprotein. The splicing, i.e., intron removal, occurs at a defined splicesite, e.g., typically at least about 4 nucleotides, between cDNA andintron sequence. For example, without limitation, the sense andantisense intron segments illustrated herein, which form adouble-stranded RNA contained no splice sites.

Isogenic: organisms (e.g., plants), which are genetically identical,except that they may differ by the presence or absence of a heterologousDNA sequence.

Isolated: The term “isolated” as used herein means that a material hasbeen removed by the hand of man and exists apart from its original,native environment and is therefore not a product of nature. An isolatedmaterial or molecule (such as a DNA molecule or enzyme) may exist in apurified form or may exist in a non-native environment such as, forexample, in a transgenic host cell. For example, a naturally occurringpolynucleotide or polypeptide present in a living animal is notisolated, but the same polynucleotide or polypeptide, separated fromsome or all of the coexisting materials in the natural system, isisolated. Such polynucleotides can be part of a vector and/or suchpolynucleotides or polypeptides could be part of a composition, andwould be isolated in that such a vector or composition is not part ofits original environment. Preferably, the term “isolated” when used inrelation to a nucleic acid, as in “an isolated nucleic acid sequence”refers to a nucleic acid sequence that is identified and separated fromat least one contaminant nucleic acid with which it is ordinarilyassociated in its natural source. Isolated nucleic acid is nucleic acidpresent in a form or setting that is different from that in which it isfound in nature. In contrast, non-isolated nucleic acids are nucleicacids such as DNA and RNA, which are found in the state they exist innature. For example, a given DNA sequence (e.g., a gene) is found on thehost cell chromosome in proximity to neighboring genes; RNA sequences,such as a specific mRNA sequence encoding a specific protein, are foundin the cell as a mixture with numerous other mRNAs, which encode amultitude of proteins. However, an isolated nucleic acid sequencecomprising for example SEQ ID NO: 1 includes, by way of example, suchnucleic acid sequences in cells which ordinarily contain SEQ ID NO:1where the nucleic acid sequence is in a chromosomal or extrachromosomallocation different from that of natural cells, or is otherwise flankedby a different nucleic acid sequence than that found in nature. Theisolated nucleic acid sequence may be present in single-stranded ordouble-stranded form. When an isolated nucleic acid sequence is to beutilized to express a protein, the nucleic acid sequence will contain ata minimum at least a portion of the sense or coding strand (i.e., thenucleic acid sequence may be single-stranded). Alternatively, it maycontain both the sense and antisense strands (i.e., the nucleic acidsequence may be double-stranded).

Mammal: The terms “mammal” or “mammalian” are intended to encompasstheir normal meaning. While the invention is most desirably intended forefficacy in humans, it may also be employed in domestic mammals such ascanines, felines, and equines, as well as in mammals of particularinterest, e.g., zoo animals, farmstock and the like.

Mature protein: protein which is normally targeted to a cellularorganelle, such as a chloroplast, and from which the transit peptide hasbeen removed.

Minimal Promoter: promoter elements, particularly a TATA element, thatare inactive or that have greatly reduced promoter activity in theabsence of upstream activation. In the presence of a suitabletranscription factor, the minimal promoter functions to permittranscription.

Non-coding: The term “non-coding” refers to sequences of nucleic acidmolecules that do not encode part or all of an expressed protein.Non-coding sequences include but are not limited to introns, promoterregions, 3′ untranslated regions, and 5′ untranslated regions.

Nucleic acids and nucleotides: The terms “Nucleic Acids” and“Nucleotides” refer to naturally occurring or synthetic or artificialnucleic acid or nucleotides. The terms “nucleic acids” and “nucleotides”comprise deoxyribonucleotides or ribonucleotides or any nucleotideanalogue and polymers or hybrids thereof in either single- ordouble-stranded, sense or antisense form. Unless otherwise indicated, aparticular nucleic acid sequence also implicitly encompassesconservatively modified variants thereof (e.g., degenerate codonsubstitutions) and complementary sequences, as well as the sequenceexplicitly indicated. The term “nucleic acid” is used inter-changeablyherein with “gene”, “cDNA, “mRNA”, “oligonucleotide,” and“polynucleotide”. Nucleotide analogues include nucleotides havingmodifications in the chemical structure of the base, sugar and/orphosphate, including, but not limited to, 5-position pyrimidinemodifications, 8-position purine modifications, modifications atcytosine exocyclic amines, substitution of 5-bromo-uracil, and the like;and 2′-position sugar modifications, including but not limited to,sugar-modified ribonucleotides in which the 2′-OH is replaced by a groupselected from H, OR, R, halo, SH, SR, NH2, NHR, NR2, or CN. shRNAs alsocan comprise non-natural elements such as non-natural bases, e.g.,ionosin and xanthine, non-natural sugars, e.g., 2′-methoxy ribose, ornon-natural phosphodiester linkages, e.g., methylphosphonates,phosphorothioates and peptides.

Nucleic acid sequence: The phrase “nucleic acid sequence” refers to asingle or double-stranded polymer of deoxyribonucleotide orribonucleotide bases read from the 5′- to the 3′-end. It includeschromosomal DNA, self-replicating plasmids, infectious polymers of DNAor RNA and DNA or RNA that performs a primarily structural role.“Nucleic acid sequence” also refers to a consecutive list ofabbreviations, letters, characters or words, which representnucleotides. In one embodiment, a nucleic acid can be a “probe” which isa relatively short nucleic acid, usually less than 100 nucleotides inlength. Often a nucleic acid probe is from about 50 nucleotides inlength to about 10 nucleotides in length. A “target region” of a nucleicacid is a portion of a nucleic acid that is identified to be ofinterest. A “coding region” of a nucleic acid is the portion of thenucleic acid, which is transcribed and translated in a sequence-specificmanner to produce into a particular polypeptide or protein when placedunder the control of appropriate regulatory sequences. The coding regionis said to encode such a polypeptide or protein.

Nucleotide sequence of interest: The term “nucleotide sequence ofinterest” refers to any nucleotide sequence, the manipulation of whichmay be deemed desirable for any reason (e.g., confer improvedqualities), by one of ordinary skill in the art. Such nucleotidesequences include, but are not limited to, coding sequences ofstructural genes (e.g., reporter genes, selection marker genes, drugresistance genes, growth factors, etc.), and non-coding regulatorysequences which do not encode an mRNA or protein product, (e.g.,promoter sequence, polyadenylation sequence, termination sequence,enhancer sequence, etc.). A nucleic acid sequence of interest maypreferably encode for an agronomically valuable trait.

Oligonucleotide: The term “oligonucleotide” refers to an oligomer orpolymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) ormimetics thereof, as well as oligonucleotides havingnon-naturally-occurring portions which function similarly. Such modifiedor substituted oligonucleotides are often preferred over native formsbecause of desirable properties such as, for example, enhanced cellularuptake, enhanced affinity for nucleic acid target and increasedstability in the presence of nucleases. An oligonucleotide preferablyincludes two or more nucleomonomers covalently coupled to each other bylinkages (e.g., phosphodiesters) or substitute linkages.

Operable linkage: The term “operable linkage” or “operably linked” is tobe understood as meaning, for example, the sequential arrangement of aregulatory element (e.g. a promoter) with a nucleic acid sequence to beexpressed and, if appropriate, further regulatory elements (such ase.g., a terminator) in such a way that each of the regulatory elementscan fulfill its intended function to allow, modify, facilitate orotherwise influence expression of said nucleic acid sequence. Theexpression may result depending on the arrangement of the nucleic acidsequences in relation to sense or antisense RNA. To this end, directlinkage in the chemical sense is not necessarily required. Geneticcontrol sequences such as, for example, enhancer sequences, can alsoexert their function on the target sequence from positions which arefurther away, or indeed from other DNA molecules. Preferred arrangementsare those in which the nucleic acid sequence to be expressedrecombinantly is positioned behind the sequence acting as promoter, sothat the two sequences are linked covalently to each other. The distancebetween the promoter sequence and the nucleic acid sequence to beexpressed recombinantly is preferably less than 200 base pairs,especially preferably less than 100 base pairs, very especiallypreferably less than 50 base pairs. In a preferred embodiment, thenucleic acid sequence to be transcribed is located behind the promoterin such a way that the transcription start is identical with the desiredbeginning of the chimeric RNA of the invention. Operable linkage, and anexpression construct, can be generated by means of customaryrecombination and cloning techniques as described (e.g., in Maniatis T,Fritsch E F and Sambrook J (1989) Molecular Cloning: A LaboratoryManual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor(N.Y.); Silhavy et al. (1984) Experiments with Gene Fusions, Cold SpringHarbor Laboratory, Cold Spring Harbor (N.Y.); Ausubel et al. (1987)Current Protocols in Molecular Biology, Greene Publishing Assoc. andWiley Interscience; Gelvin et al. (Eds) (1990) Plant Molecular BiologyManual; Kluwer Academic Publisher, Dordrecht, The Netherlands). However,further sequences, which, for example, act as a linker with specifiscleavage sites for restriction enzymes, or as a signal peptide, may alsobe positioned between the two sequences. The insertion of sequences mayalso lead to the expression of fusion proteins. Preferably, theexpression construct, consisting of a linkage of promoter and nucleicacid sequence to be expressed, can exist in a vector-integrated form andbe inserted into a plant genome, for example by transformation.

Organ: The term “organ” with respect to a plant (or “plant organ”) meansparts of a plant and may include (but shall not limited to) for exampleroots, fruits, shoots, stem, leaves, anthers, sepals, petals, pollen,seeds, etc. The term “organ” with respect to an animal (“animal organ”)means parts of an animal and may include (but shall not limited to) forexample external organs (such as arms, legs, head, etc.) or internalorgans (such as heart, kidney, liver, stomach, etc.).

Overhang: An “overhang” is a relatively short single-stranded nucleotidesequence on the 5′- or 3′-hydroxyl end of a double-strandedoligonucleotide molecule (also referred to as an “extension,”“protruding end,” or “sticky end”).

Plant: The terms “plant” or “plant organism” refer to any organism,which is capable of photosynthesis, and the cells, tissues, parts orpropagation material (such as seeds or fruits) derived therefrom.Encompassed within the scope of the invention are all genera and speciesof higher and lower plants of the Plant Kingdom. Annual, perennial,monocotyledonous and dicotyledonous plants and gymnosperms arepreferred. A “plant” refers to any plant or part of a plant at any stageof development. Mature plants refer to plants at any developmental stagebeyond the seedling stage. Encompassed are mature plant, seed, shootsand seedlings, and parts, propagation material (for example tubers,seeds or fruits) and cultures, for example cell cultures or calluscultures,) derived therefrom. Seedling refers to a young, immature plantat an early developmental stage. Therein are also included cuttings,cell or tissue cultures and seeds. As used in conjunction with thepresent invention, the term “plant tissue” includes, but is not limitedto, whole plants, plant cells, plant organs, plant seeds, protoplasts,callus, cell cultures, and any groups of plant cells organized intostructural and/or functional units. Preferably, the term “plant” as usedherein refers to a plurality of plant cells, which are largelydifferentiated into a structure that is present at any stage of aplant's development. Such structures include one or more plant organsincluding, but are not limited to, fruit, shoot, stem, leaf, flowerpetal, etc. More preferably, the term “plant” includes whole plants,shoot vegetative organs/structures (e.g. leaves, stems and tubers),roots, flowers and floral organs/structures (e.g. bracts, sepals,petals, stamens, carpels, anthers and ovules), seeds (including embryo,endosperm, and seed coat) and fruits (the mature ovary), plant tissues(e.g. vascular tissue, ground tissue, and the like) and cells (e.g.guard cells, egg cells, trichomes and the like), and progeny of same.The class of plants that can be used in the method of the invention isgenerally as broad as the class of higher and lower plants amenable totransformation techniques, including angiosperms (monocotyledonous anddicotyledonous plants), gymnosperms, ferns, and multicellular algae. Itincludes plants of a variety of ploidy levels, including aneuploid,polyploid, diploid, haploid and hemizygous. Included within the scope ofthe invention are all genera and species of higher and lower plants ofthe plant kingdom. Included are furthermore the mature plants, seed,shoots and seedlings, and parts, propagation material (for example seedsand fruit) and cultures, for example cell cultures, derived therefrom.Preferred are plants and plant materials of the following plantfamilies: Amaranthaceae, Brassicaceae, Carophyllaceae, Chenopodiaceae,Compositae, Cucurbitaceae, Labiatae, Leguminosae, Papilionoideae,Liliaceae, Linaceae, Malvaceae, Rosaceae, Saxifragaceae,Scrophulariaceae, Solanaceae, Tetragoniaceae. Annual, perennial,monocotyledonous and dicotyledonous plants are preferred host organismsfor the generation of transgenic plants. The use of the recombinationsystem, or method according to the invention is furthermore advantageousin all ornamental plants, forestry, fruit, or ornamental trees, flowers,cut flowers, shrubs or turf. Said plant may include—but shall not belimited to—bryophytes such as, for example, Hepaticae (hepaticas) andMusci (mosses); pteridophytes such as ferns, horsetail and clubmosses;gymnosperms such as conifers, cycads, ginkgo and Gnetaeae; algae such asChlorophyceae, Phaeophpyceae, Rhodophyceae, Myxophyceae, Xanthophyceae,Bacillariophyceae (diatoms) and Euglenophyceae. Plants for the purposesof the invention may comprise the families of the Rosaceae such as rose,Ericaceae such as rhododendrons and azaleas, Euphorbiaceae such aspoinsettias and croton, Caryophyllaceae such as pinks, Solanaceae suchas petunias, Gesneriaceae such as African violet, Balsaminaceae such astouch-me-not, Orchidaceae such as orchids, Iridaceae such as gladioli,iris, freesia and crocus, Compositae such as marigold, Geraniaceae suchas geraniums, Liliaceae such as Drachaena, Moraceae such as ficus,Araceae such as philodendron and many others. The transgenic plantsaccording to the invention are furthermore selected in particular fromamong dicotyledonous crop plants such as, for example, from the familiesof the Leguminosae such as pea, alfalfa and soybean; the family of theUmbelliferae, particularly the genus Daucus (very particularly thespecies carota (carrot)) and Apium (very particularly the speciesgraveolens var. dulce (celery)) and many others; the family of theSolanaceae, particularly the genus Lycopersicon, very particularly thespecies esculentum (tomato) and the genus Solanum, very particularly thespecies tuberosum (potato) and melongena (aubergine), tobacco and manyothers; and the genus Capsicum, very particularly the species annum(pepper) and many others; the family of the Leguminosae, particularlythe genus Glycine, very particularly the species max (soybean) and manyothers; and the family of the Cruciferae, particularly the genusBrassica, very particularly the species napus (oilseed rape), campestris(beet), oleracea cv Tastie (cabbage), oleracea cv Snowball Y(cauliflower) and oleracea cv Emperor (broccoli); and the genusArabidopsis, very particularly the species thaliana and many others; thefamily of the Compositae, particularly the genus Lactuca, veryparticularly the species sativa (lettuce) and many others. Thetransgenic plants according to the invention are selected in particularamong monocotyledonous crop plants, such as, for example, cereals suchas wheat, barley, sorghum and millet, rye, triticale, maize, rice oroats, and sugarcane. Further preferred are trees such as apple, pear,quince, plum, cherry, peach, nectarine, apricot, papaya, mango, andother woody species including coniferous and deciduous trees such aspoplar, pine, sequoia, cedar, oak, etc. Especially preferred areArabidopsis thaliana, Nicotiana tabacum, oilseed rape, soybean, corn(maize), wheat, linseed, potato and tagetes.

Polynucleotide construct. The term “polynucleotide construct” refers toa nucleic acid at least partly created by recombinant methods. The term“DNA construct” is referring to a polynucleotide construct consisting ofdeoxyribonucleotides. The construct may be single- or—preferably—doublestranded. The construct may be circular or linear. The skilled worker isfamiliar with a variety of ways to obtain one of a DNA construct.Constructs can be prepared by means of customary recombination andcloning techniques as are described, for example, in Maniatis T, FritschE F and Sambrook J (1989) Molecular Cloning: A Laboratory Manual, 2ndEd., Cold Spring Harbor Laboratory, Cold Spring Harbor (N.Y.); Silhavyet al. (1984) Experiments with Gene Fusions, Cold Spring HarborLaboratory, Cold Spring Harbor (N.Y.); Ausubel et al. (1987) CurrentProtocols in Molecular Biology, Greene Publishing Assoc. and WileyInterscience; Gelvin et al. (Eds) (1990) Plant Molecular Biology Manual;Kluwer Academic Pub-lisher, Dordrecht, The Netherlands.

Polypeptide: The terms “polypeptide”, “peptide”, “oligopeptide”,“polypeptide”, “gene product”, “expression product” and “protein” areused interchangeably herein to refer to a polymer or oligomer ofconsecutive amino acid residues.

Pre-protein: Protein, which is normally targeted to a cellularorganelle, such as a chloroplast, and still comprising its transitpeptide.

Promoter: The terms “promoter,” “promoter element,” or “promotersequence” are equivalents and as used herein, refers to a DNA sequencewhich when ligated to a nucleotide sequence of interest is capable ofcontrolling the transcription of the nucleotide sequence of interestinto mRNA. A promoter is typically, though not necessarily, located 5′(i.e., up-stream) of a nucleotide sequence of interest (e.g., proximalto the transcriptional start site of a structural gene) whosetranscription into mRNA it controls, and provides a site for specificbinding by RNA polymerase and other transcription factors for initiationof transcription. A polynucleotide sequence is “heterologous to” anorganism or a second polynucleotide sequence if it originates from aforeign species, or, if from the same species, is modified from itsoriginal form. For example, a promoter operably linked to a heterologouscoding sequence refers to a coding sequence from a species differentfrom that from which the promoter was derived, or, if from the samespecies, a coding sequence which is not naturally associated with thepromoter (e.g. a genetically engineered coding sequence or an allelefrom a different ecotype or variety). Suitable promoters can be derivedfrom genes of the host cells where expression should occur or frompathogens for this host cells (e.g., plants or plant pathogens likeplant viruses). If a promoter is an inducible promoter, then the rate oftranscription increases in response to an inducing agent. In contrast,the rate of transcription is not regulated by an inducing agent if thepromoter is a constitutive promoter. Also, the promoter may be regulatedin a tissue-specific or tissue preferred manner such that it is onlyactive in transcribing the associated coding region in a specific tissuetype(s) such as leaves, roots or meristem. The term “tissue specific” asit applies to a promoter refers to a promoter that is capable ofdirecting selective expression of a nucleotide sequence of interest to aspecific type of tissue (e.g., petals) in the relative absence ofexpression of the same nucleotide sequence of interest in a differenttype of tissue (e.g., roots). Tissue specificity of a promoter may beevaluated by, for example, operably linking a reporter gene to thepromoter sequence to generate a reporter construct, introducing thereporter construct into the genome of a plant such that the reporterconstruct is integrated into every tissue of the resulting transgenicplant, and detecting the expression of the reporter gene (e.g.,detecting mRNA, protein, or the activity of a protein encoded by thereporter gene) in different tissues of the transgenic plant. Thedetection of a greater level of expression of the reporter gene in oneor more tissues relative to the level of expression of the reporter genein other tissues shows that the promoter is specific for the tissues inwhich greater levels of expression are detected. The term “cell typespecific” as applied to a promoter refers to a promoter, which iscapable of directing selective expression of a nucleotide sequence ofinterest in a specific type of cell in the relative absence ofexpression of the same nucleotide sequence of interest in a differenttype of cell within the same tissue. The term “cell type specific” whenapplied to a promoter also means a promoter capable of promotingselective expression of a nucleotide sequence of interest in a regionwithin a single tissue. Cell type specificity of a promoter may beassessed using methods well known in the art, e.g., GUS activitystaining or immunohistochemical staining. The term “constitutive” whenmade in reference to a promoter means that the promoter is capable ofdirecting transcription of an operably linked nucleic acid sequence inthe absence of a stimulus (e.g., heat shock, chemicals, light, etc.).Typically, constitutive promoters are capable of directing expression ofa transgene in substantially any cell and any tissue. In contrast, a“regulatable” promoter is one which is capable of directing a level oftranscription of an operably linked nuclei acid sequence in the presenceof a stimulus (e.g., heat shock, chemicals, light, etc.) which isdifferent from the level of transcription of the operably linked nucleicacid sequence in the absence of the stimulus.

Purified: As used herein, the term “purified” refers to molecules,either nucleic or amino acid sequences that are removed from theirnatural environment, isolated or separated. “Substantially purified”molecules are at least 60% free, preferably at least 75% free, and morepreferably at least 90% free from other components with which they arenaturally associated. An purified nucleic acid sequence may be anisolated nucleic acid sequence.

Recombinant: The term “recombinant” with respect to polypeptides orproteins refer to polypeptides or proteins produced by recombinant DNAtechniques, i.e., produced from cells transformed by an exogenousrecombinant DNA construct encoding the desired polypeptide or protein.Recombinant nucleic acids and polypeptide may also comprise molecules,which as such does not exist in nature but are modified, changed,mutated or otherwise manipulated by man. Preferably, a “recombinantpolypeptide” is a non-naturally occurring polypeptide that differs insequence from a naturally occurring polypeptide by at least one aminoacid residue. Preferred methods for producing said recombinantpolypeptide and/or nucleic acid may comprise directed or non-directedmutagenesis, DNA shuffling or other methods of recursive recombination.

Sense: The term “sense” is understood to mean a nucleic acid having asequence which is homologous or identical to a target sequence, forexample a sequence which binds to a protein transcription factor andwhich is involved in the expression of a given gene. According to apreferred embodiment, the nucleic acid comprises a gene of interest andelements allowing the expression of the said gene of interest.

Significant Increase or Decrease: An increase or decrease, for examplein enzymatic activity or in gene expression, that is larger than themargin of error inherent in the measurement technique, preferably anincrease or decrease by about 2-fold or greater of the activity of thecontrol enzyme or expression in the control cell, more preferably anincrease or decrease by about 5-fold or greater, and most preferably anincrease or decrease by about 10-fold or greater.

Stabilize: To “stabilize” the expression of a nucleotide sequence in aplant cell means that the level of expression of the nucleotide sequenceafter applying a method of the present invention is approximately thesame in cells from the same tissue in different plants from the samegeneration or throughout multiple generations when the plants are grownunder the same or comparable conditions.

Substantially complementary: In its broadest sense, the term“substantially complementary”, when used herein with respect to anucleotide sequence in relation to a reference or target nucleotidesequence, means a nucleotide sequence having a percentage of identitybetween the substantially complementary nucleotide sequence and theexact complementary sequence of said reference or target nucleotidesequence of at least 60%, more desirably at least 70%, more desirably atleast 80% or 85%, preferably at least 90%, more preferably at least 93%,still more preferably at least 95% or 96%, yet still more preferably atleast 97% or 98%, yet still more preferably at least 99% or mostpreferably 100% (the later being equivalent to the term “identical” inthis context). Preferably identity is assessed over a length of at least19 nucleotides, preferably at least 50 nucleotides, more preferably theentire length of the nucleic acid sequence to said reference sequence(if not specified otherwise below). Sequence comparisons are carried outusing default GAP analysis with the University of Wisconsin GCG, SEQWEBapplication of GAP, based on the algorithm of Needleman and Wunsch(Needleman and Wunsch (1970) J Mol. Biol. 48: 443-453; as definedabove). A nucleotide sequence “substantially complementary” to areference nucleotide sequence hybridizes to the reference nucleotidesequence under low stringency conditions, preferably medium stringencyconditions, most preferably high stringency conditions (as definedabove).

Substantially identical: In its broadest sense, the term “substantiallyidentical”, when used herein with respect to a nucleotide sequence,means a nucleotide sequence corresponding to a reference or targetnucleotide sequence, wherein the percentage of identity between thesubstantially identical nucleotide sequence and the reference or targetnucleotide sequence is desirably at least 60%, more desirably at least70%, more desirably at least 80% or 85%, preferably at least 90%, morepreferably at least 93%, still more preferably at least 95% or 96%, yetstill more preferably at least 97% or 98%, yet still more preferably atleast 99% or most preferably 100% (the later being equivalent to theterm “identical” in this context). Preferably identity is assessed overa length of at least 19 nucleotides, preferably at least 50 nucleotides,more preferably the entire length of the nucleic acid sequence to saidreference sequence (if not specified otherwise below). Sequencecomparisons are carried out using default GAP analysis with theUniversity of Wisconsin GCG, SEQWEB application of GAP, based on thealgorithm of Needleman and Wunsch (Needleman and Wunsch (1970) J Mol.Biol. 48: 443-453; as defined above). A nucleotide sequence“substantially identical” to a reference nucleotide sequence hybridizesto the exact complementary sequence of the reference nucleotide sequence(i.e. its corresponding strand in a double-stranded molecule) under lowstringency conditions, preferably medium stringency conditions, mostpreferably high stringency conditions (as defined above). Homologes of aspecific nucleotide sequence include nucleotide sequences that encode anamino acid sequence that is at least 24% identical, more preferably atleast 35% identical, yet more preferably at least 50% identical, yetmore preferably at least 65% identical to the reference amino acidsequence, as measured using the parameters described above, wherein theamino acid sequence encoded by the homolog has the same biologicalactivity as the protein encoded by the specific nucleotide. The term“substantially identical”, when used herein with respect to apolypeptide, means a protein corresponding to a reference polypeptide,wherein the polypeptide has substantially the same structure andfunction as the reference protein, e.g. where only changes in aminoacids sequence not affecting the polypeptide function occur. When usedfor a polypeptide or an amino acid sequence the percentage of identitybetween the substantially similar and the reference polypeptide or aminoacid sequence desirably is at least 24%, more desirably at least 30%,more desirably at least 45%, preferably at least 60%, more preferably atleast 75%, still more preferably at least 90%, yet still more preferablyat least 95%, yet still more preferably at least 99%, using default GAPanalysis parameters as described above. Homologes are amino acidsequences that are at least 24% identical, more preferably at least 35%identical, yet more preferably at least 50% identical, yet morepreferably at least 65% identical to the reference polypeptide or aminoacid sequence, as measured using the parameters described above, whereinthe amino acid sequence encoded by the homolog has the same biologicalactivity as the reference polypeptide.

Synthetic: As used herein, “synthetic” means made wholly by chemicalmeans, e.g. through the annealing of chemically-synthesizedcomplementary oligonucleotides rather than by biological means, e.g.through the amplification of a chemically-synthesized template using thepolymerase chain reaction (PCR) or other enzyme-mediated biologicalreactions such as ligation or phosphorylation. In preferred embodiments,the oligonucleotides are synthesized using commercial oligonucleotidesynthesis machines, including but not limited to the ABI 394 and ABI3900 DNA/RNA Synthesizers available from Applied Biosystems, Inc. orother commercially-equivalent synthesizers.

Target gene: The terms “target”, “target gene” and “target nucleotidesequence” are used equivalently. As used herein, a target gene can beany gene of interest present in an eukaryotic organism (such as aplant). A target gene may be endogenous or introduced. For example, atarget gene is a gene of known function or is a gene whose function isunknown, but whose total or partial nucleotide sequence is known.Alternatively, the function of a target gene and its nucleotide sequenceare both unknown. A target gene is a native gene of the eukaryotic cellor is a heterologous gene which has previously been introduced into theeukaryotic cell or a parent cell of said eukaryotic cell, for example bygenetic transformation. A heterologous target gene is stably integratedin the genome of the eukaryotic cell or is present in the eukaryoticcell as an extrachromosomal molecule, e.g. as an autonomouslyreplicating extrachromosomal molecule. A target gene may includepolynucleotides comprising a region that encodes a polypeptide orpolynucleotide region that regulates replication, transcription,translation, or other process important in expression of the targetprotein; or a polynucleotide comprising a region that encodes the targetpolypeptide and a region that regulates expression of the targetpolypeptide; or noncoding regions such as the 5′ or 3′ UTR or introns. Atarget gene may refer to, for example, an mRNA molecule produced bytranscription a gene of interest. Furthermore, the term “correspond,” asin “an chimeric RNA comprising a sequence that corresponds to a targetgene sequence,” means that the two sequences are complementary orhomologous or bear such other biologically rational relationship to eachother (e.g., based on the sequence of nucleomonomers and theirbase-pairing properties). The “target gene” to which an chimeric RNAmolecule of the invention is directed may be associated with apathological condition. For example, the gene may be apathogen-associated gene, e.g., a viral gene, a tumor-associated gene, adefective gene (e.g., an abnormal cancer-causing gene), or an autoimmunedisease-associated gene. The target gene may also be a heterologous geneexpressed in a recombinant cell or a genetically altered organism. Bydetermining or modulating (e.g., inhibiting) the function of such agene, valuable information and therapeutic benefits in medicine,veterinary medicine, and biology may be obtained.

Tissue: The term “tissue” with respect to an organism (e.g., a plant;“plant tissue”) means arrangement of multiple cells includingdifferentiated and undifferentiated tissues of the organism. Tissues mayconstitute part of an organ (e.g., the epidermis of a plant leaf or ananimal skin) but may also constitute tumor tissues (e.g., callus tissue)and various types of cells in culture (e.g., single cells, protoplasts,embryos, calli, protocorm-like bodies, etc.). The tissue may be in vivo(e.g., in planta), in organ culture, tissue culture, or cell culture.

Transformation: The term “transformation” as used herein refers to theintroduction of genetic material (e.g., a transgene or heterologousnucleic acid molecules) into a cell, tissue or organism. Transformationof a cell may be stable or transient. The term “transienttransformation” or “transiently transformed” refers to the introductionof one or more transgenes into a cell in the absence of integration ofthe transgene into the host cell's genome. Transient transformation maybe detected by, for example, enzyme-linked immunosorbent assay (ELISA),which detects the presence of a polypeptide encoded by one or more ofthe transgenes. Alternatively, transient transformation may be detectedby detecting the activity of the protein (e.g., β-glucuronidase) encodedby the transgene (e.g., the uid A gene). The term “transienttransformant” refers to a cell which has transiently incorporated one ormore transgenes. In contrast, the term “stable transformation” or“stably transformed” refers to the introduction and integration of oneor more transgenes into the genome of a cell, preferably resulting inchromosomal integration and stable heritability through meiosis. Stabletransformation of a cell may be detected by Southern blot hybridizationof genomic DNA of the cell with nucleic acid sequences, which arecapable of binding to one or more of the transgenes. Alternatively,stable transformation of a cell may also be detected by the polymerasechain reaction of genomic DNA of the cell to amplify transgenesequences. The term “stable transformant” refers to a cell, which hasstably integrated one or more transgenes into the genomic DNA. Thus, astable transformant is distinguished from a transient transformant inthat, whereas genomic DNA from the stable transformant contains one ormore transgenes, genomic DNA from the transient transformant does notcontain a transgene. Transformation also includes introduction ofgenetic material into plant cells in the form of plant viral vectorsinvolving epichromosomal replication and gene expression, which mayexhibit variable properties with respect to meiotic stability.Transformed cells, tissues, or plants are understood to encompass notonly the end product of a transformation process, but also transgenicprogeny thereof.

Transgene: The term “transgene” as used herein refers to any nucleicacid sequence, which is introduced into the genome of a cell byexperimental manipulations. A transgene may be an “endogenous DNAsequence,” or a “heterologous DNA sequence” (i.e., “foreign DNA”). Theterm “endogenous DNA sequence” refers to a nucleotide sequence, which isnaturally found in the cell into which it is introduced so long as itdoes not contain some modification (e.g., a point mutation, the presenceof a selectable marker gene, etc.) relative to the naturally-occurringsequence.

Transgenic: The term transgenic when referring to a cell, tissue ororganisms means transformed, preferably stably transformed, with arecombinant DNA molecule that preferably comprises a suitable promoteroperatively linked to a DNA sequence of interest.

Unaffected: As used herein, “essentially unaffected” refers to a levelof an agent such as a protein or mRNA transcript that is either notaltered by a particular event or altered only to an extent that does notaffect the physiological function of that agent. In a preferred aspect,the level of the agent that is essentially unaffected is within 20%,more preferably within 10%, and even more preferably within 5% of thelevel at which it is found in a cell or organism that lacks a nucleicacid molecule capable of selectively reducing another agent. As usedherein, “substantially unaffected” refers to a level of an agent such asa protein or mRNA transcript in which the level of the agent that issubstantially unaffected is within 49%, more preferably within 35%, andeven more preferably within 24% of the level at which it is found in acell or organism that lacks a nucleic acid molecule capable ofselectively reducing another agent. As used herein, “partiallyunaffected” refers to a level of an agent such as a protein or mRNAtranscript in which the level of the agent that is partially unaffectedis within 80%, more preferably within 65%, and even more preferablywithin 50% of the level at which it is found in a cell or organism thatlacks a nucleic acid molecule capable of selectively reducing anotheragent.

Vector: As used herein, the term “vector” refers to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked. One type of vector is a genomic integrated vector, or“integrated vector”, which can become integrated into the chromosomalDNA of the host cell. Another type of vector is an episomal vector,i.e., a nucleic acid capable of extra-chromosomal replication. Vectorscapable of directing the expression of genes to which they areoperatively linked are referred to herein as “expression vectors”. Inthe present specification, “plasmid” and “vector” are usedinterchangeably unless otherwise clear from the context. Expressionvectors designed to produce RNAs as described herein in vitro or in vivomay contain sequences under the controt of any RNA polymerase, includingmitochondrial RNA polymerase, RNA pol I, RNA pol II, and RNA pol III.These vectors can be used to transcribe the desired RNA molecule in thecell according to this invention. Vectors may be desirably designed toutilize an endogenous mitochondrial RNA polymerase (e.g., humanmitochondrial RNA polymerase, in which case such vectors may utilize thecorresponding human mitochondrial promoter). Mitochondrial polymerasesmay be used to generate capped (through expression of a capping enzyme)or uncapped messages in vivo. RNA pol I, RNA pol II, and RNA pol IIItranscripts may also be generated in vivo. Such RNAs may be capped ornot, and if desired, cytoplasmic capping may be accomplished by variousmeans including use of a capping enzyme such as a vaccinia cappingenzyme or an alphavirus capping enzyme. The DNA vector is designed tocontain one of the promoters or multiple promoters in combination(mitochondrial, RNA poll, II, or pollIII, or viral, bacterial orbacteriophage promoters along with the cognate polymerases). Preferably,where the promoter is RNA pol II, the sequence encoding the RNA moleculehas an open reading frame greater than about 300 nts to avoiddegradation in the nucleus. Such plasmids or vectors can include plasmidsequences from bacteria, viruses or phages. Such vectors includechromosomal, episomal and virus-derived vectors e.g., vectors derivedfrom bacterial plasmids, bacteriophages, yeast episomes, yeastchromosomal elements, and viruses, vectors derived from combinationsthereof, such as those derived from plasmid and bacteriophage geneticelements, cosmids and phagemids. Thus, one exemplary vector is a singleor double-stranded phage vector. Another exemplary vector is a single ordouble-stranded RNA or DNA viral vector. Such vectors may be introducedinto cells as polynucleotides, preferably DNA, by well known techniquesfor introducing DNA and RNA into cells. The vectors, in the case ofphage and viral vectors may also be and preferably are introduced intocells as packaged or encapsidated virus by well known techniques forinfection and transduction. Viral vectors may be replication competentor replication defective. In the latter case, viral propagationgenerally occurs only in complementing host cells.

Wild-type: The term “wild-type”, “natural” or of “natural origin” meanswith respect to an organism, polypeptide, or nucleic acid sequence, thatsaid organism is naturally occurring or available in at least onenaturally occurring organism which is not changed, mutated, or otherwisemanipulated by man.

DETAILED DESCRIPTION OF THE INVENTION

A first embodiment of the invention relates to a method for transgenicexpression with enhanced specificity in an eukaryotic organism saidmethod comprising the steps of:

-   a) providing an expression construct comprising a promoter sequence    functional in said eukaryotic organism and functionally linked    thereto a nucleotide sequence to be expressed into a chimeric RNA    sequence, said nucleotide sequence comprising    -   i) at least one sequence capable to confer a preferred phenotype        or beneficial effect to said eukaryotic organism, and    -   ii) at least one sequence substantially complementary to a        microRNA sequence naturally expressed in said eukaryotic        organism, wherein said microRNA is naturally expressed in        tissues, at times, and/or under environmental conditions, where        expression is not desired, but is not or substantially less        expressed in tissues, at times, and/or under environmental        conditions, where such expression is desired,    -   wherein at least one of sequence i) and sequence ii) are        heterologous to each other, and-   b) introducing said expression construct into a eukaryotic organism.

Preferably, said eukaryotic organism is a human, an animal or a plant.

It is not unusual that some ‘tissue-specific’ promoters having leakinessof expression in other tissues which could result in undesirablephenotype such as phytotoxicity. In other cases, it has been proved verychallenge to generate tissue-specific promoter for certain application(e.g., ‘syncytium-specific’ promoters for achieving nematode resistancein plants). Given that some miRNAs have tissues-specific and/or temporalexpression pattern, one could design a generic vector with a miRNA-tag(a short sequence substantially complementary or complementary to agiven endogenous miRNA) at 3′UTR of an expression construct (e.g.,comprised in a binary vector), so that leakiness of transgene expressionin the tissues where miRNA are expressed will be reduced or eliminated.

The essential, inventive feature of the invention disclosed herein isthe incorporation of “at least one sequence substantially complementaryto a microRNA sequence naturally expressed in said eukaryotic organism”(i.e. the target organism, where the enhanced expression specificityshould be achieved). Said sequence—hereinafter also the “microRNAtag”—suppress or lower expression or will lead to enhanced degradation(thereby suppressing or lowering expression) of the chimeric RNAsequence in tissues, at times, and/or under environmental conditionswhere the endogenous miRNA is expressed.

Without being limited to any specific functional mechanism of action,the endogenous miRNA is thought to interact with the miRNA-tag in thechimeric RNA sequence, thereby inducing its degradation (or genesilencing). This silencing is surprisingly found to be restricted to thetissue, time, and/or under environmental condition where the endogenousmiRNA is naturally expressed and is found not to spread over the entireorganism.

1. The miRNA-tag of the Invention

1.1 General Properties

The miRNA-tag a sequence, which is substantially complementary to amicroRNA (miRNA) sequence naturally expressed in an eukaryotic organism(i.e. an endogenous miRNA). The terms naturally occurring miRNA (ormicroRNA) and endogenous miRNA (or micro RNA) have the same meaning andare used interchangeable herein.

The miRNA-tags of the invention are complementary or substantiallycomplementary to an endogenous miRNA. While the invention does notdepend on miRNA-tags of a particular size, the miRNA-tags will have alength similar to the length of the endogenous miRNAs, such miRNAs knownin the art typically comprise between about 15 and 30 nucleotides. Thus,the miRNA-tag will preferably be a small sequence comprising about 15 toabout 30 nucleotides, about 20 to about 28 nucleotides, morespecifically about 21-24 nucleotides. Generally the miRNA-tag will becompletely complementary to the endogenous miRNA, however, mismatchesmay be tolerated, thus it is contemplated that the miRNA-tag issubstantially complementary to the miRNA naturally expressed in aneukaryotic organism. The term substantially complementary as used inthis context (i.e. for the complementarity between the miRNA-tag and anendogenous miRNA) means, that generally from 1 to about 6 mismatches mayoccur, more specifically about 2 to 3 mismatched nucleotides may beincluded in the miRNA-tag in comparison to the endogenous miRNAsequence. Alternatively, the complement of the miRNA-tag may have andidentity to the sequence of the endogenous miRNA of at least 60% or 70%,preferably at least 80% or 85%, more preferably at least 90%, mostpreferably at least 95%. While the mismatched nucleotides may occurthroughout the miRNA sequence (i.e. in any position), preferably, theyare located in the region near or in the 3′ region of the endogenousmiRNA. The 3′-region of the endogenous miRNA is complementary to the5′-region of the miRNA tag. Accordingly, said mismatches are preferablyin the 5′-region of the miRNA-tag. It has been demonstrated, that forexample, 3 mismatches plus a G::U wobble can be engineered at 3′ regionof miRNA without affecting its function (Mallory et al., EMBO Journal,23:3356-3364, (2004)). Accordingly, in the most preferred embodiment theterm substantially complement means that 3.5 mismatches (i.e. 3 truemismatches plus one G:U wobble counted as 0.5) can occur between themiRNA-tag and the endogenous miRNA. In this manner, a miRNA sequence canbe designed to modulate the expression of any target sequence.

1.2 Identification of Suitable miRNAs for Designing miRNA Tags

To allow for enhanced expression specificity, the microRNA (to which thesequence comprised in the nucleotide sequence to be expressed issubstantially complementary) is preferably not constitutively expressed,but is varying in expression in at least one parameter selected from thegroup consisting of tissue, special, time, development, environmental orother exogenous factors. Preferably, the microRNA is tissue-specificor—preferentially expressed, spatially-regulated, developmentalregulated, and/or regulated by other factors such as biotic or abioticstress factors.

A tissue-tissue specific—or preferentially expressed miRNA is understoodherein as an miRNA which is not expressed to the same extent in alltissues of an organism at a given specific time (such expression profilemay or may not change over time (e.g., during development or aging) orunder other conditions (exogenous factors such as stress). Preferably,the miRNA is expressed only in one or a few tissues, while it is notexpressed to a significant amount (e.g., an amount which is readilydetectable by standard RNA detection methods such as Northern blot) inother tissues.

A miRNA regulated by other factors may include miRNAs which are up- ordownregulated (in one, more or all tissues) upon interaction of theorganism with a factor, preferably an exogenous factor, more preferablya stress stimuli. Such stress stimuli may comprise abiotic and bioticstress factors. Given the fact that maize miR160 (see Examples fordetails) is a stress-induced microRNA, it is very possible that someother miRNAs are induced by a range of environmental stimuli (e.g.biotic stress, and chemicals). Using similar strategies proposed above,one can control transgene expression in response to environmentalstimuli in certain tissues.

There are several approaches to identify and isolate miRNAs in variousorganism and tissues. For example, after total RNA is isolated from anorganism or specific tissues or cell types, RNA is resolved on adenaturing 15% polyacrylamide gel. A gel fragment represents the sizerange of 15 to 26 nucleotides is excised, small RNA is eluted, andrecovered. Subsequently, small RNA is ligated to 5′ and 3′ RNA/DNAchimeric oligonucleotide adapters. Reverse transcription reaction isperformed using RT primer followed by PCR with appropriate primers. PCRproducts are then cloned into vector for sequencing (Sunkar R and Zhu JK, The Plant Cell 16:2001:2019, 2004) Several other techniques andmethods have been applied to detect miRNA in an organism or tissues suchas Northern blot analysis, ribonucleases protection-based PAGE,microarray-based miRNA profiling and qRT-PCR Tagman analysis.

There are various ways to “design” a miRNA-tag to achieve a certainexpression profile. For example, first, one chooses miRNA expressed inthe tissue(s) (or at times or under conditions) where there is leakyexpression of gene-of-interest, which should be prevented. Second, onedetermines complementary sequence of miRNA and insert such shortnucleotide sequences into the gene-of-interest (e.g., the 5′UTR region,3′ UTR region, or even the coding region without affecting the functionof gene-of-interest).

1.2 Localization within the Expressed Chimeric RNA

Various positions are possible for the sequence being substantiallycomplementary to the microRNA (hereinafter also the “microRNA tag”) inthe nucleotide sequence to be expressed. Preferably, the sequence beingsubstantially complementary to the microRNA is positioned in a locationof the nucleotide sequence to be expressed corresponding to the5′-untranslated region or the 3′-untranslated region of said sequence

1.3 Production and/or Expression of the Chimeric RNA of the Invention

The term “chimeric RNA” or “chimeric RNA molecule” or “chimericribonucleotide sequence” are used interchangeable herein and areintended to mean an polynucleotide molecule, which is at least in partconsisting of ribonucleotides, which comprises

-   i) at least one sequence substantially complementary to a microRNA    sequence naturally occurring in a eukaryotic organism, and-   i) at least one other sequence (preferably a sequence capable to    confer a preferred phenotype or beneficial effect to an eukaryotic    organism),    wherein at least one of sequence i) and sequence ii) are    heterologous to each other (i.e. are not covalently linked in nature    or in an natural (i.e. non-genetically modified) organism or cell).

The fact the chimeric RNA sequence of the invention is “at least in partconsisting of ribonucleotides” means—for example—that the chimeric RNAsequence may comprise other than ribonucleotide bases. As describedbelow, the chimeric RNA molecule of the invention may also be obtainedby chemically synthesis. By this method, other than natural occurringribonucleotide residues (e.g., modified residues) may be incorporated).

The chimeric RNA molecules expressed by the method of the invention(i.e. the RNA comprising the miRNA-tag) are as such considered to benovel and inventive. Not only there expression constructs can be used,but also the chimeric RNA molecules as such has strong potential forindustrial applicability, especially in the field of pharmaceuticalapplication, where activity of a RNA-based pharmaceutical is sought toact only on certain tissue, at certain times or under certainconditions.

Thus anther embodiment of the invention relates to a chimericribonucleotide sequence comprising

-   i) at least one sequence capable to confer a preferred phenotype or    beneficial effect to a eukaryotic organism, and-   ii) at least one sequence substantially complementary to a microRNA    sequence naturally occurring in a eukaryotic organism,    wherein at least one of sequence i) and sequence ii) are    heterologous to each other.

The sequences i) and/or ii) in said chimeric ribonucleotide sequence arepreferably defined as for the method of the invention.

The chimeric RNA molecule (i.e. the RNA molecule comprising the miRNAtag) can be produced and applied to the host cell or organism by variousmeans, familiar to the person skilled in the art. The chimeric RNAmolecules of the invention can be produced or synthesized by any methodknown in the art, e.g., using recombinant expression, enzymaticsynthesis or chemical synthesis. The RNA molecules can be synthesized invitro (e.g., using enzymatic synthesis and chemical synthesis) or invivo (using recombinant DNA technology well known in the art).

For example, the chimeric RNA may be produced outside the eukaryotictarget cell or may be produced recombinantly (e.g., by an expressionconstruct) within the target cell. In one embodiment, the chimeric RNAmolecule of the invention can be produced by enzymatic synthetic methodsor chemical synthetic methods in vitro. In another embodiment, thechimeric RNA molecule may be generated in a recombinant culture, e.g.,bacterial cells, isolated therefrom, and used in the methods discussedbelow. In another embodiment another agent (such as an expressionconstruct or vector) generates the chimeric RNA molecule in vivo afterdelivery to the target cell or organism. The target cell or organism ispreferably a mammalian, plant cell or animal (such as a nematode) cellor organism.

For example the chimeric RNA molecule can be

-   a) expressed from an expression construct or an expression vector in    the target cell or organism, or-   b) expressed from an expression construct in an in vivo or in vitro    transcription system, wherein the chimeric RNA molecule is purified    from said transcription system and introduced into the host cell or    organism (e.g., by feeding or injection), or-   c) chemical synthesis of the chimeric RNA molecule introduced into    the host cell or organism (e.g., by feeding or injection).

1.3.1 Expression of the Chimeric RNA by Recombinant Expression

The chimeric RNA molecule of the invention can be made by recombinantexpression. Thus, in one embodiment of the invention the chimeric RNA isproduced in the cell by an expression construct or expression vector.The chimeric RNA molecule can be made (e.g., expressed) directly in theeukaryotic target cell or organism, where it can directly fulfill itsfunction without the need of further introduction. Alternatively thechimeric RNA molecule can be expressed in another cell, optionallypurified, and subsequently delivered into the target cell or organism.Thus, the RNA molecule of this invention can be made in a recombinantmicroorganism, e.g., bacteria and yeast or in a recombinant host cell ororganism, e.g., plant or mammalian cells, and—optionally—isolated fromthe cultures thereof by conventional techniques. See, e.g., thetechniques described in Sambrook et al, MOLECULAR CLONING, A LABORATORYMANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N. Y., 1989, which is exemplary of laboratory manuals thatdetail these techniques, and the techniques described in U.S. Pat. Nos.5,824,538; 5,877,159 and 65,643,771, incorporated herein by reference.

Where the RNA molecules of the invention are formed in vivo they arepreferably produced employing an expression construct or expressionvector. More preferably the expression construct or vector is comprisinga nucleic acid sequence, preferably a double stranded DNA molecule,encoding at least one of the above-described chimeric RNA molecules ofthe invention, operably linked to a transcription regulating sequence (apromoter) which is capable to realize transcription of said nucleic acidsequence in the chosen host or target cell to produce a chimeric RNA ofthe invention. As discussed, a number of promoters can be used in thepractice of the invention. The promoters can be selected based on thedesired outcome. Thus, the nucleotide sequence for expression of thechimeric RNA can be combined with constitutive, tissue-preferred,inducible, developmental, or other promoters for expression in plantsdepending upon the desired outcome. Specific promoters are describedbelow.

Such expression constructs for expression of said chimericribonucleotide sequence (which are employed in the method of theinvention) are considered to be novel and inventive. Thus anotherembodiment of the invention relates to an expression constructcomprising a promoter sequence functional in a eukaryotic organism andfunctionally linked thereto a nucleotide sequence to be expressed, saidsequence comprising

-   i) at least one sequence capable to confer a preferred phenotype or    beneficial effect to said eukaryotic organism, and-   ii) at least one sequence substantially complementary to a microRNA    sequence naturally occurring in said eukaryotic organism,    wherein at least one of sequence i) and sequence ii) are    heterologous to each other.

The expression construct and its elements are preferably defined asabove for the method of the invention.

Another embodiment of the invention relates to an expression vectorcomprising an expression construct of the invention. Preferably, theexpression vector is an eukaryotic expression vector. More preferablythe eukaryotic expression vector is a viral vector, a plasmid vector ora binary vector.

The use and production of an expression construct are known in the art(see also WO 97/32016; U.S. Pat. Nos. 5,593,874, 5,698,425, 5,712,135,5,789,214, and 5,804,693; and the references cited therein).

For transcription from a transgene in vivo or an expression construct, aregulatory region (e.g., promoter, enhancer, silencer, splice donor andacceptor, polyadenylation) may be used to transcribe the chimeric RNA.Transcription may be targeted by specific transcription in an organ,tissue, or cell type; stimulation of an environmental condition (e.g.,infection, stress, temperature, chemical inducers); and/or engineeringtranscription at a developmental stage or age. The RNA strands may ormay not be polyadenylated; the RNA strands may or may not be capable ofbeing translated into a polypeptide by a cell's translational apparatus.Various promoters can be used for expression of the nucleotide sequencecomprising the microRNA-tag. The promoters can—for example—be selectedfrom the group consisting of constitutive promoters, tissue-specific ortissue-preferential promoters, and inducible promoters. A tissuespecific promoter in this context, does—preferably—mean which is leaky(i.e. having expression activity in other than the preferred or maintissue) to a small but measurable extent. More specific examples forpreferred expression constructs are described below for the specificapplication.

The nucleotide sequence to be expressed to form a chimeric RNA moleculemay have various form and/or functions. For example, it may comprise anopen reading frame encoding a protein. Alternatively, it may encode afunctional RNA selected from the group consisting of antisense RNA,sense RNA, double-stranded RNA or ribozymes. Said functional RNA ispreferably attenuating expression of an endogenous gene. For expressionof a function RNA, it is desirable that the sequences, which enableprotein expression, e.g., Kozak regions, etc., are not included in theseexpression constructs of the invention. The expression construct for theexpression of the nucleotide sequence comprising the microRNA-tag can beDNA, RNA and can be single- or double-stranded. Preferably theexpression construct is DNA, more preferably double-stranded DNA. Theexpression construct can be part or a larger vector construct.Preferably, the expression construct is in a plasmid. The expressionconstruct is preferably comprised in an expression vector. Thus anotherembodiment of the invention relates to an expression vector comprisingan expression construct of the invention. The expression vector can be aDNA or RNA molecule, can be single stranded or double stranded, can be aplasmid or other type of vector (as defined above and specified for thevarious application and technical field below in detail). Morepreferably the expression vector is a double-stranded, circular plasmidDNA vector. A further embodiment of the invention relates to anexpression vector comprising an expression construct of the invention.Examples of vectors (see above in the DEFINITION section for details)can be plasmids, cosmids, phages, viruses or else Agrobacteria.Preferably, the vector is a eukaryotic expression vector. Morepreferably, the eukaryotic expression vector is a viral vector orplasmid vector. In certain embodiments, the expression constructs orvectors are episomal, e.g., and transfection is transient. In otherembodiments, the expression constructs or vectors are chromosomallyintegrated, e.g., to produce a stably transfected cell line. Preferredvectors for forming such stable cell lines are described in U.S. Pat.No. 6,025,192 and WO/9812339, which are incorporated by referenceherein. Vectors for expression in E. coli are preferably pQE70, pQE60and pQE-9 (QIAGEN, Inc.); pBluescript vectors, Phagescript vectors,pNH8A, pNH16a, pNH18A, pNH46A (Stratagene Cloning Systems, Inc.);ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia Biotech, Inc.).

As described above (and for specific organisms and cells below in moredetail), the expression construct and vector may be introduced intoorganisms or cells. Yet another embodiment of the invention relates to atransformed cell or non-human organism comprising an expressionconstruct or an expression vector of the invention. Preferably, saidexpression construct or expression vector is inserted into the genome(preferably the chromosomal or plastid DNA) of said cell or organism.Preferably, said cell or organism is selected from the group ofmammalian, bacterial, fungal, nematode or plant cells and organism.Another embodiment of the invention relates to tissues, part andpropagation material of the transformed organism of the invention. Incase of transformed plants the propagation material is preferablytransformed seed.

The expression construct can be inserted into the vector (preferably aplasmid vector) via a suitable restriction cleavage site. The resultingvector is first introduced into E. coli. Correctly transformed E. coliare selected, grown, and the recombinant vector is obtained by methodswith which the skilled worker is familiar. Restriction analysis andsequencing can be employed for verifying the cloning step. Preferredvectors are those, which make possible a stable integration of theexpression construct into the host genome. Suitable promoters and vectorconstructs are described in United States Patent Application No.20040220130.

The vectors designed to produce the chimeric RNA of the invention maydesirably be designed to generate two or more, including a number ofdifferent chimeric RNAs. This approach is desirable in that a singlevector may produce many, independently operative chimeric RNAs ratherthan a single chimeric RNA molecule from a single transcription unit andby producing a multiplicity of different chimeric RNAs. Various meansmay be employed to achieve this, including autocatalytic sequences aswell as sequences for cleavage to create random and/or predeterminedsplice sites.

The construction of polynucleotide constructs generally requires the useof vectors able to replicate in bacteria. A plethora of kits arecommercially available for the purification of plasmids from bacteria.For their proper use, follow the manufacturers instructions (see, forexample, EasyPrep™, FlexiPrep™, both from Pharmacia Biotech;StrataClean™, from Stratagene; and, QIAprep™, Qiagen). The isolated andpurified plasmids can then be further manipulated to produce otherplasmids, used to transfect cells or incorporated into other vectorsystems (e.g., Agrobacterium tumefaciens) to infect and transform targetcells or organism (preferably plants).

Still other suitable vector (or delivery agents) for introducing achimeric RNA of the invention into a target cell include live,attenuated or killed, inactivated viruses, and particularly recombinantviruses carrying the required RNA polynucleotide sequence discussedabove. Such viruses may be designed similarly to recombinant virusespresently used to deliver genes to cells for gene therapy and the like,but preferably do not have the ability to express a protein orfunctional fragment of a protein. Among useful viruses or viralsequences which may be manipulated to provide the required RNA moleculeto the mammalian cell in vivo are, without limitation, alphavirus,adenovirus, adeno-associated virus, baculoviruses, delta virus, poxviruses, hepatitis viruses, herpes viruses, papova viruses (such asSV40), poliovirus, pseudorabies viruses, retroviruses, vaccinia viruses,positive and negative stranded RNA viruses, viroids, and virusoids, orportions thereof. These various viral delivery agents may be designed byapplying conventional techniques such as described in M. Di Nocola etal, Cancer Gene Ther., 5(6):350-6 (1998), among others, with theteachings of the present invention. A viral construct packaged into aviral particle would accomplish both efficient introduction of anexpression construct into the cell and transcription of chimeric RNAconstruct encoded by the expression construct.

Another delivery agent for providing the chimeric RNA molecules of theinvention in the target cell or organism include live, attenuated orkilled, inactivated donor cells which have been transfected or infectedin vitro with a synthetic RNA molecule or an expression construct orvector as described above. These donor cells may then be administered orfeed to the target organism (e.g., a mammal or a pathogen such as anematode), as described in detail below, to stimulate the mechanism inthe target organism which mediates this inhibitory effect. These donorcells are desirably eukaryotic cells, such as mammalian cells C127, 3T3,CHO, HeLa, human kidney 293, BHK cell lines, and COS-7 cells, andpreferably are of the same mammalian species as the mammalian recipient,or plant cells. Such donor cells can be made using techniques similar tothose described in, e.g., Emerich et al, J. Neurosci., 16: 5168-81(1996). Even more preferred, the donor cells may be harvested from thespecific mammal to be treated and made into donor cells by ex vivomanipulation, akin to adoptive transfer techniques, such as thosedescribed in D. B. Kohn et al, Nature Med. 4(7):775-80 (1998). Donorcells may also be from non-mammalian species, if desired.

1.3.2 Production of the Chimeric RNA of the Invention by EnzymaticSynthesis

The chimeric RNA molecule according to this invention may be deliveredto the target cell or organism as a molecule, which was made in vitro byenzymatic synthesis.

Thus, another embodiment of the invention relates to a method forgenerating a chimeric RNA of the invention comprising:

-   (i) providing an in vitro transcription system including an    expression construct for the chimeric RNA of the invention, and-   (ii) isolating said chimeric RNA of the invention.

Prokaryotic and—preferably—eukaryotic transcription systems can beemployed. Furthermore, systems based on isolated enzymes and systemsbased on cellular extracts can be utilized. Eukaryotic, prokaryotic orbacteriophage RNA polymerases (such as, for example, T3, T7 or SP6 RNApolymerase) can be used for this purpose. Suitable methods for thein-vitro expression of RNA are described (WO 97/32016; U.S. Pat. Nos.5,593,874; 5,698,425, 5,712,135, 5,789,214, 5,804,693). Enzymaticsystems based on isolated enzymes can be used, for example, thebacteriophage T7, T3 or SP6 RNA polymerases according to theconventional methods described by such texts as the Promega Protocolsand Applications Guide, (3rd ed. 1996), eds. Doyle, ISBN No.1-882274-57-1.

Accordingly, the invention also provides a kit that includes reagentsfor attenuating the expression of a target gene in a cell. The kitcontains a DNA template comprising a promoter (preferably a T7 promoter,a T3 promoter or an SP6 promoter) operably linked to a nucleotidesequence encoding a chimeric RNA of the invention. The kit optionallycontains amplification primers for amplifying the DNA sequence from theDNA template and nucleotide triphosphates (i.e., ATP, GTP, CTP and UTP)for forming RNA. Also optionally, the kit contains a RNA polymerase,capable of binding to the promoter on the DNA template and causingtranscription of the nucleotide sequence to which the promoter isoperably linked; a purification column for purifying single strandedRNA, such as a size exclusion column; one or more buffers, for example abuffer for annealing single stranded RNAs to yield double stranded RNA;and RNAse A or RNAse T for purifying double stranded RNA.

In cases where an eukaryotic transcription system is employed (such aslysates from rabbit reticulocytes or wheat germ; see Movahedzadeh etal., “In vitro transcription and translation,” in Methods in MolecularBiology, V. 235, N. Casali, A. Preston, Eds., Totowa, N.J.: HumanaPress, p. 247-55; Lamla et al., Acta Biochim Pol, 48:453-65, 2001)correct removal of the removable RNA element is expected resulting inrelease of the chimeric RNA, which may be purified from the system.

Prior to introduction into a cell, tissue or organism, a chimeric RNAwhich has been synthesized in vitro, either chemically or enzymatically,can be purified either completely or in part from the reaction mixture,for example by extraction, precipitation, electrophoresis,chromatography or combinations of these methods.

1.3.3 Production of the Chimeric RNA of the Invention by ChemicalSynthesis

The chimeric RNA molecules of the invention can also besynthesized—entirely or in part—by chemical synthesis. Chemicalsynthesis of linear oligonucleotides is well known in the art and can beachieved by solution or solid phase techniques. Preferably, synthesis isby solid phase methods. Suitable synthetic procedures include but arenot limited to phosphoramidite, phosphite triester, H-phosphonate, andphosphotriester methods, typically by automated synthesis methods. Sucholigonucleotide synthesis protocols can be found, e.g., in U.S. Pat. No.5,830,653; WO 98/13526; Stec et al. 1984. J. Am. Chem. Soc. 106:6077;Stec et al. 1985. J. Org. Chem. 50:3908; Stec et al. J. Chromatog. 1985.326:263; LaPlanche et al. 1986. Nuc. Acid. Res. 1986. 14:9081; Fasman G.D., 1989. Practical Handbook of Biochemistry and Molecular Biology.1989. CRC Press, Boca Raton, Fla.; Lamone. 1993. Biochem. Soc. Trans.21:1; U.S. Pat. Nos. 5,013,830; 5,214,135; 5,525,719; Kawasaki et al.1993. J. Med. Chem. 36:831; WO 92/03568; U.S. Pat. Nos. 5,276,019;5,264,423. Alternative methods for in vitro chemical synthetis of theRNA molecules of the invention are described [see, e.g., Xu et al, Nucl.Acids Res., 24(18):3643-4 (1996); Naryshkin et al, Bioorg. Khim.,22(9):691-8 (1996); Grasby et al, Nucl. Acids Res., 21(19):4444-50(1993); Chaix el al, Nucl. Acids Res., 17(18):7381-93 (1989); Chou etal, Biochem., 2(6):2422-35 (1989); Odai et al, Nucl. Acids Symp, Ser.,21:105-6 (1989); Naryshkin et al, Bioorg. Khim, 22(9):691-8 (1996); Sunet al, RNA, 3(11):1352-1363 (1997); X. Zhang et al, Nucl. Acids Res.,25(20):3980-3 (1997); Grvaznov et al, Nucl. Acids Res., 26 (18):4160-7(1998); Kadokura et al, Nucl. Acids Symp Ser, 37:77-8 (1997); Davison etal, Biomed. Pept. Proteins, Nucl. Acids, 2(1):1-6 (1996); Mudrakovskaiaet al, Bioorg. Khim., 17(6):819-22 (1991)].

The synthesis method selected can depend on the length of the desiredoligonucleotide and such choice is within the skill of the ordinaryartisan. For example, the phosphoramidite and phosphite triester methodcan produce oligonucleotides having 175 or more nucleotides while theH-phosphonate method works well for oligonucleotides of less than 100nucleotides. If modified bases are incorporated into theoligonucleotide, and particularly if modified phosphodiester linkagesare used, then the synthetic procedures are altered as needed accordingto known procedures. In this regard, Uhlmann et al. (1990, ChemicalReviews 90:543-584) provide references and outline procedures for makingoligonucleotides with modified bases and modified phosphodiesterlinkages. Other exemplary methods for making oligonucleotides are taughtin Sonveaux. 1994. “Protecting Groups in Oligonucleotide Synthesis”;Agrawal. Methods in Molecular Biology 26:1. Exemplary synthesis methodsare also taught in “Oligonucleotide Synthesis—A Practical Approach”(Gait, M. J. IRL Press at Oxford University Press. 1984). Moreover,linear oligonucleotides of defined sequence, including some sequenceswith modified nucleotides, are readily available from several commercialsources.

The chimeric RNA molecule of the invention may include modifications toeither the phosphate-sugar backbone or the nucleoside. For example, thephosphodiester linkages of natural RNA may be modified to include atleast one of a nitrogen or sulfur heteroatom. Modifications in RNAstructure may be tailored to allow specific genetic inhibition whileavoiding a general panic response in some organisms by dsRNA. Likewise,bases may be modified to block the activity of adenosine deaminase. Inone embodiment of the invention the chimeric RNA molecule has end-blockson one or both ends.

The chimeric RNA of the invention may include “morpholinooligonucleotides.” Morpholino oligonucleotides are non-ionic andfunction by an RNase H-independent mechanism. Each of the 4 geneticbases (Adenine, Cytosine, Guanine, and Thymine/Uracil) of the morpholinooligonucleotides is linked to a 6-membered morpholine ring. Morpholinooligonucleotides are made by joining the 4 different subunit types by,e.g., non-ionic phosphorodiamidate inter-subunit linkages. Morpholinooligonucleotides have many advantages including: complete resistance tonucleases (Antisense & Nuc. Acid Drug Dev. 1996. 6:267); predictabletargeting (Biochemica Biophysica Acta. 1999. 1489:141); reliableactivity in cells (Antisense & Nuc. Acid Drug Dev. 1997. 7:63);excellent sequence specificity (Antisense & Nuc. Acid Drug Dev. 1997.7:151); minimal non-antisense activity (Biochemica Biophysica Acta.1999. 1489:141); and simple osmotic or scrape delivery (Antisense & Nuc.Acid Drug Dev. 1997. 7:291). Morpholino oligonucleotides are alsopreferred because of their non-toxicity at high doses. A discussion ofthe preparation of morpholino oligonucleotides can be found in Antisense& Nuc. Acid Drug Dev. 1997. 7:187.

Another embodiment of the invention includes duplexes in whichnucleomonomernucleomonomer mismatches are present in a sense 2′-O-methlystrand (and are thought to be easier to unwind). As a further example,the use of 2′-O-methyl RNA may beneficially be used in circumstances inwhich it is desirable to minimize cellular stress responses. RNA having2′-O-methyl nucleomonomers may not be recognized by cellular machinerythat is thought to recognize unmodified RNA. The use of 2′-O-methylatedor partially 2′-O-methylated RNA may avoid the interferon response todouble-stranded nucleic acids, while maintaining target RNA inhibition.This RNA interference (“stealth RNAi”) is useful for avoiding theinterferon or other cellular stress responses, both in short RNAi (e.g.,siRNA) sequences that induce the interferon response, and in longer RNAisequences that may induce the interferon response. Other chemicalmodifications in addition to 2′-O-methylation may also achieve thiseffect.

In certain embodiments, the chimeric RNA molecules of the inventioncomprise 3′ and 5′ termini (except for circular molecules). In oneembodiment, the 3′ and 5′ termini can be substantially protected fromnucleases e.g., by modifying the 3′ or 5′ linkages (e.g., U.S. Pat. No.5,849,902 and WO 98/13526). For example, oligonucleotides can be maderesistant by the inclusion of a “blocking group.” The term “blockinggroup” as used herein refers to substituents (e.g., other than OHgroups) that can be attached to oligonucleotides or nucleomonomers,either as protecting groups or coupling groups for synthesis (e.g.,FITC, propyl, phosphate, hydrogen phosphonate, or phosphoramidite).“Blocking groups” also include “end blocking groups” or “exonucleaseblocking groups” which protect the 5′ and 3′ termini of theoligonucleotide, including modified nucleotides and non-nucleotideexonuclease resistant structures. Exemplary end-blocking groups includecap structures (e.g., a 7-methylguanosine cap), inverted nucleomonomers,e.g., with 3′-3′ or 5′-5′ end inversions (see, e.g., Ortiagao et al.1992. Antisense Res. Dev. 2:129), methylphosphonate, phosphoramidite,non-nucleotide groups (e.g., non-nucleotide linkers, amino linkers,conjugates) and the like. The 3′ terminal nucleomonomer can comprise amodified sugar moiety. The 3′ terminal nucleomonomer comprises a 3′-Othat can optionally be substituted by a blocking group that prevents3′-exonuclease degradation of the oligonucleotide. For example, the3′-hydroxyl can be esterified to a nucleotide through a 3′-3′internucleotide linkage. For example, the alkyloxy radical can bemethoxy, ethoxy, or isopropoxy, and preferably, ethoxy. Optionally, the3′-3′ linked nucleotide at the 3′ terminus can be linked by a substitutelinkage. To reduce nuclease degradation, the 5′ most 3′-5′ linkage canbe a modified linkage, e.g., a phosphorothioate or aP-alkyloxyphosphotriester linkage. Optionally, the 5′ terminal hydroxymoiety can be esterified with a phosphorus containing moiety, e.g.,phosphate, phosphorothioate, or P-ethoxyphosphate.

In another embodiment of the invention the chimeric RNA molecule of theinvention may comprise one or more flexible linker. Such linkers may beused to combine or fuse two or more smaller chimeric RNAs together to alarger chimeric RNA molecule. A linker is provided with functionalgroups at each end that can be suitably protected or activated. Thefunctional groups are covalently attached to each RNA molecule, e.g.,via an ether, ester, carbamate, phosphate ester or amine linkage toeither the 5′-hydroxyl or the 3′-hydroxyl. Preferred linkages arephosphate ester linkages similar to typical oligonucleotide linkages.For example, hexaethyleneglycol can be protected on one terminus with aphotolabile protecting group (i.e., NVOC or MeNPOC) and activated on theother terminus with 2-cyanoethyl-N,N-diisopropylamino-chlorophosphite toform a phosphoramidite. Other methods of forming ether, carbamate oramine linkages are known to those of skill in the art and particularreagents and references can be found in such texts as March, AdvancedOrganic Chemistry, 4th Ed., Wiley-Interscience, New York, N.Y., 1992. Ingeneral, the flexible linkers are non-nucleotide molecules includingspacers, attachments, bioconjugates, chromophores, reporter groups, dyelabeled RNAs, and non-naturally occurring nucleotide analogues.Preferred linkers, spacers, bioconjugates, attachments, and chromophoresare more specifically described in US Patent Application No.20040058886, herein incorporated by reference.

In one embodiment, a chimeric RNA molecule of the invention, which issought to function in gene silencing, can include an agent whichincreases the affinity for its target sequence. The term “affinityenhancing agent” includes agents that increase the affinity of anchimeric RNA molecule of the invention for its target. Such agentsinclude, e.g., intercalating agents and high affinity nucleomonomers.Intercalating agents interact strongly and nonspecifically with nucleicacids. Intercalating agents serve to stabilize RNA-DNA duplexes and thusincrease the affinity of the chimeric RNA molecule of the invention fortheir targets. Intercalating agents are most commonly linked to the 3′or 5′ end of oligonucleotides. Examples of intercalating agents includeacridine, chlorambucil, benzopyridoquinoxaline, benzopyridoindole,benzophenanthridine, and phenazinium. The agents may also impart othercharacteristics to the oligonucleotide, for example, increasingresistance to endonucleases and exonucleases.

In one embodiment, a high affinity nucleomonomer is incorporated into anchimeric RNA molecule of the invention. The language “high affinitynucleomonomer” as used herein includes modified bases or base analogsthat bind to a complementary base in a target nucleic acid molecule withhigher affinity than an unmodified base, for example, by having moreenergetically favorable interactions with the complementary base, e.g.,by forming more hydrogen bonds with the complementary base. For example,high affinity nucleomonomer analogs such as aminoethyoxy phenoxazine(also referred to as a G clamp), which forms four hydrogen bonds withguanine are included in the term “high affinity nucleomonomer.” A highaffinity nucleomonomer is illustrated below (see, e.g., Flanagan, etal., 1999. Proc. Natl. Acad. Sci. 96:3513). Other exemplary highaffinity nucleomonomers are known in the art and include 7-alkenyl,7-alkynyl, 7-heteroaromatic-, or 7-alkynylheteroaromatic-substitutedbases or the like which can be substituted for adenosine or guanosine inoligonucleotides (see, e.g., U.S. Pat. No. 5,594,121). Also,7-substituted deazapurines have been found to impart enhanced bindingproperties to oligonucleotides, i.e., by allowing them to bind withhigher affinity to complementary target nucleic acid molecules ascompared to unmodified oligonucleotides. High affinity nucleomonomerscan be incorporated into the oligonucleotides of the instant inventionusing standard techniques.

In another embodiment, an agent that increases the affinity of achimeric RNA molecule of the invention for its target comprises anintercalating agent. As used herein, the language “intercalating agent”includes agents which can bind to a DNA double helix. When covalentlyattached to a chimeric RNA molecule of the invention, an intercalatingagent enhances the binding of the oligonucleotide to its complementarygenomic DNA target sequence. The intercalating agent may also increaseresistance to endonucleases and exonucleases. Exemplary intercalatingagents are taught by Helene and Thuong (1989. Genome 31:413), andinclude e.g., acridine derivatives (Lacoste et al. 1997. Nucleic AcidsResearch. 25:1991; Kukreti et al. 1997. Nucleic Acids Research.25:4264); quinoline derivatives (Wilson et al. 1993. Biochemistry32:10614); benzo[f]quino[3,4-b]quioxaline derivatives (Marchand et al.1996. Biochemistry. 35:5022; Escude et al. 1998. Proc. Natl. Acad. Sci.95:3591). Intercalating agents can be incorporated into a chimeric RNAmolecule of the invention using any convenient linkage. For example,acridine or psoralen can be linked to the oligonucleotide through anyavailable —OH or —SH group, e.g., at the terminal 5′ position of theoligonucleotide, the 2′ positions of sugar moieties, or an OH, NH2,COOH, or SH incorporated into the 5-position of pyrimidines usingstandard methods.

In one embodiment, the double-stranded duplex constructs of theinvention can be further stabilized against nucleases by forming loopstructures at the 5′ or 3′ end of the sense or antisense strand of theconstruct. Suitable loop-structure and other structures to stabilize anRNA molecule of the invention are for example described in US patentApplication No. 20040014956.

The chimeric RNA molecule of the invention (or an expression constructor vector for its production) can be derivatized, chemically modified,combined with and/or linked to various agents to enhance its activity orspecificity. Such agents include but are not limited to conjugationagents (e.g., for improvement of cellular uptake), protein carriers(e.g., for improvement of cellular uptake and greater cellularaccumulation), encapsulating agents (such as liposomes; e.g., tofacilitate the cellular uptake or targeting), complexing agents (such ascationic lipid, e.g., to increase cellular uptake), basic oligopeptides,transporting peptides (e.g., HIV TAT transcription factor, lactoferrin,Herpes VP22 protein), and targeting agents (for targeting to a cellularreceptor). Suitable conjugation agents, protein carriers, encapsulatingagents, complexing agents, basic oligopeptides, transporting peptides,and targeting agents are described for example in US Patent ApplicationNo. 20040014956. Additional ways to contact a chimeric RNA molecule ofthe invention with its target cell are described below in the context ofpharmaceutical application. Alternatively, the chimeric RNA can bedelivered to the target organism by ingestion or infection of atransgenic organism comprising an expression construct for the chimericRNA. See e.g., U.S. Pat. No. 6,506,559. Methods for increase stabilityof the RNA molecules of the invention against nuclease degradation(e.g., by serum nucleases and cellular nucleases and nucleases found inother bodily fluids) are described in United States Patent ApplicationNo. 20040014956.

If synthesized chemically or by in vitro enzymatic synthesis, thechimeric RNA molecule of the invention may be purified prior tointroduction into the cell. For example, RNA can be purified from amixture by extraction with a solvent or resin, precipitation,electrophoresis (e.g., polyacrylamide gel electrophoresis),chromatography (e.g., gel chromatography and high pressure liquidchromatography) or a combination thereof. Alternatively, the chimericRNA may be used with no or a minimum of purification to avoid losses dueto sample processing. The chimeric RNA may be dried for storage ordissolved in an aqueous solution. The quality of the synthesizedchimeric RNA molecules of the invention synthesized can be verified bycapillary electrophoresis and denaturing strong anion HPLC (SAX-HPLC)using, e.g., the method of Bergot and Egan. 1992. J. Chrom. 599:35.

1.4 Introduction of the Chimeric RNA into Cells and Organism

The chimeric RNA of the invention or its delivery or production agents(e.g., expression constructs or vectors) (hereinafter together the “RNAagent”) can be introduced into an organism or a cell in various wayswith which the skilled worker is familiar. “To introduce” is to beunderstood in the broad sense and comprises, for the purposes of thepresent invention, all those methods which are suitable for directly orindirectly introducing, into an organism or a cell, compartment, tissue,organ or seed of same, a RNA agent of the invention, or generatingit/them therein. The introduction can bring about the transient presenceof a RNA agent, or else a stable presence.

Thus a further aspect of the invention relates to cells and organism(e.g., plant, animal, protozoan, virus, bacterium, or fungus), whichcomprise at least one chimeric RNA of the invention, or an RNA agent(e.g., an expression construct or expression vectors encoding saidchimeric RNA molecule). In certain embodiments, the cell is suspended inculture; while in other embodiments the cell is in (or part of) a wholeorganism (e.g., a microorganism, plant or an animal, such as a non-humanmammal). The cell can be prokaryotic or of eukaryotic nature.Preferably, the expression construct is comprised with the genomic DNA,more preferably within the chromosomal or plastidic DNA, most preferablyin the chromosomal DNA of the cell.

The cell having the target gene may be from the germ line or somatic,totipotent or pluripotent, dividing or non-dividing, parenchyma orepithelium, immortalized or transformed, or the like. The cell can be agamete or an embryo; if an embryo, it can be a single cell embryo or aconstituent cell or cells from a multicellular embryo. The term “embryo”thus also includes fetal tissue. The cell having the target gene may bean undifferentiated cell, such as a stem cell, or a differentiated cell,such as from a cell of an organ or tissue, including fetal tissue, orany other cell present in an organism. Cell types that aredifferentiated include adipocytes, fibroblasts, myocytes,cardiomyocytes, endothelium, neurons, glia, blood cells, megakaryocytes,lymphocytes, macrophages, neutrophils, eosinophils, basophils, mastcells, leukocytes, granulocytes, keratinocytes, chondrocytes,osteoblasts, osteoclasts, hepatocytes, and cells of the endocrine orexocrine glands.

Preferred prokaryotes are mainly bacteria such as bacteria of the genusEscherichia, Corynebacterium, Bacillus, Clostridium, Propionibacterium,Butyrivibrio, Eubacterium, Lactobacillus, Erwinia, Agrobacterium,Flavobacterium, Alcaligenes, Phaeodactylum, Colpidium, Mortierella,Entomophthora, Mucor, Crypthecodinium or Cyanobacteria, for example ofthe genus Synechocystis. Microorganisms which are preferred are mainlythose which are capable of infecting plants and thus of transferring theconstructs according to the invention. Preferred microorganisms arethose of the genus Agrobacterium and in particular the speciesAgrobacterium tumefaciens and rhizogenes.

Eukaryotic cells and organisms comprise plant and animal (preferablynonhuman) organisms and/or cells and eukaryotic microorganisms such as,for example, yeasts, algae or fungi. A corresponding transgenic organismcan be generated for example by introducing the expression systems inquestion into a zygote, stem cell, protoplast or another suitable cellwhich is derived from the organism. A transgenic animal that expresses achimeric RNA of the invention from a recombinant expression constructmay be produced by introducing the construct into a zygote, an embryonicstem cell, or another multipotent cell derived from the appropriateorganism. A viral construct packaged into a viral particle wouldaccomplish both efficient introduction of an expression construct intothe cell and transcription of RNA encoded by the expression construct.Suitable vector are will known in the art (see e.g., Shi, Y. 2003.Trends Genet 2003 January 19:9; Reichhart J M et al. Genesis. 2002.34(1-2):160-4, Yu et al. 2002. Proc Natl Acad Sci USA 99:6047; Sui etal. 2002. Proc Natl Acad Sci USA 99:5515).

The plant may be a monocot, dicot or gymnosperm; the animal may be avertebrate or invertebrate. Preferred animal and plant organisms arespecified above in the DEFINITION section. Preferred fungi areAspergillus, Trichoderma, Ashbya, Neurospora, Fusarium, Beauveria orfurther fungi described in Indian Chem Engr. Section B. Vol 37, No 1, 2(1995), page 15, Table 6. Especially preferred is the filamentousHemiascomycete Ash bya gossypii. Preferred yeasts are Candida,Saccharomyces, Hansenula or Pichia, especially preferred areSaccharomyces cerevisiae or Pichia pastoris (ATCC Accession No. 201178).Especially preferred animal organisms are nematodes.

Preferred as organisms are plant organisms. Preferred plants areselected in particular from among crop plants. Most preferred are

-   a) Plants which are suitable for oil production such as, for    example, oilseed rape, sunflower, sesame, safflower (Carthamus    tinctorius), olive tree, soybean, maize, peanut, castor-oil plant,    oil palm, wheat, cacao shrub, or various nut species such as, for    example, walnut, coconut or almond. Especially preferred among    these, in turn, are dicotyledonous plants, in particular oilseed    rape, soybean and sunflower.-   b) Plants, which serve for the production of starch, such as, for    example, maize, wheat or potato.-   c) Plants, which are used as foodstuffs and/or feeding stuffs and/or    useful plant and in which a resistance to pathogens would be    advantageous such as, for example, barley, rye, rice, potato,    cotton, flax, or linseed.-   d) Plants, which can serve for the production of fine chemicals such    as, for example, vitamins and/or carotenoids such as, for example,    oilseed rape.

Plant varieties may be excluded, particularly registrable plantvarieties according to Plant Breeders Rights. It is noted that a plantneed not be considered a “plant variety” simply because it containsstably within its genome a transgene, introduced into a cell of theplant or an ancestor thereof. In addition to a plant, the presentinvention provides any clone of such a plant, seed, selfed or hybridprogeny and descendants, and any part or propagule of any of these, suchas cuttings and seed, which may be used in reproduction or propagation,sexual or asexual. Also encompassed by the invention is a plant which isa sexually or asexually propagated off-spring, clone or descendant ofsuch a plant, or any part or propagule of said plant, off-spring, cloneor descendant. Genetically modified plants according to the invention,which can be consumed by humans or animals, can also be used as food orfeedstuffs, for example directly or following processing known in theart. The present invention also provides for parts of the organismespecially plants, particularly reproductive or storage parts. Plantparts, without limitation, include seed, endosperm, ovule, pollen,roots, tubers, stems, leaves, stalks, fruit, berries, nuts, bark, pods,seeds and flowers. In a particularly preferred embodiment of the presentinvention, the plant part is a seed.

The RNA agent (e.g., the chimeric RNA molecule of the invention) istypically is introduced or administered in an amount that allowsdelivery of at least one copy per cell. Higher amounts (for example atleast 5, 10, 100, 500 or 1000 copies per cell) can, if appropriate,affect a more efficient phenotype (e.g., higher expression or highersuppression of the target genes). The amount of RNA agent administeredto a cell, tissue, or organism depends on the nature of the cell,tissue, or organism, the nature of the target gene, and the nature ofthe RNA agent, and can readily be optimized to obtain the desired levelof expression or inhibition.

Preferably at least about 100 molecules, preferably at least about 1000,more preferably at least about 10,000 of the RNA agent, most preferablyat least about 100,000 of the RNA agent are introduced. In the case ofadministration of RNA agent to a cell culture or to cells in tissue, bymethods other than injection, for example by soaking, electroporation,or lipid-mediated transfection, the cells are preferably exposed tosimilar levels of RNA agent in the medium.

For examples the RNA agent may be introduced into cells viatransformation, transfection, injection, projection, conjugation,endocytosis, and phagocytosis. Preferred method for introductioncomprise but are not limited to:

-   a) methods of the direct or physical introduction of the chimeric    RNA molecule of the invention into the target cell or organism, and-   b) methods of the indirect introduction of chimeric RNA of the    invention into the target cell or organism (e.g., by a first    introduction of an expression construct and a subsequent    intracellular expression).

1.4.1 Direct and Physical Introduction of RNA into Target Cells orOrganism

In case the chimeric RNA of the invention (or a RNA agent) is producedoutside the target cell or organism, it can be contacted with (i.e.,brought into contact with, also referred to herein as administered ordelivered to) and taken up by one or more cell or the target organism(preferably human, pathogen or plant cells or organisms). The contactmay be in vitro, e.g., in a test tube or culture dish, (and may or maynot be introduced into a subject) or in vivo, e.g., in a subject such asa mammalian, pathogen or plant subject. The pathogen is preferably anematode.

The chimeric RNA of the invention (or a RNA agent) may be directlyintroduced into the cell (i.e., intracellularly); or introducedextracellularly into a cavity, interstitial space, into the circulationof an organism, introduced orally, or may be introduced by bathing anorganism in a solution containing the chimeric RNA of the invention (ora RNA agent). Methods for oral introduction include direct mixing of RNAwith food of the organism, as well as engineered approaches in which aspecies that is used as food is engineered to express a chimeric RNA ofthe invention (or a RNA agent), then fed to the organism to be affected.

Physical methods of introducing nucleic acids include injection of asolution of the chimeric RNA of the invention (or a RNA agent) directlyinto the cell or extracellular injection into the organism. For example,in the case of an embryo or a cell, the chimeric RNA of the invention(or a RNA agent) is conveniently administered by microinjection; othermethods of introducing nucleic acids into a cell include bombardment byparticles covered by the chimeric RNA of the invention (or a RNA agent),soaking the cell or organism in a solution of the chimeric RNA of theinvention (or a RNA agent), electroporation of cell membranes in thepresence of the chimeric RNA of the invention (or a RNA agent),liposome-mediated delivery of chimeric RNA of the invention (or a RNAagent) and transfection mediated by chemicals such as calcium phosphate.

The chimeric RNA of the invention (or a RNA agent) agent may beintroduced along with components that enhance RNA uptake by the cell, orotherwise increase its functionality. Delivery into cells can beenhanced by suitable art recognized methods including calcium phosphate,DMSO, glycerol or dextran, electroporation, or by transfection, e.g.,using cationic, anionic, or neutral lipid compositions or liposomesusing methods known in the art (see e.g., WO 90/14074; WO 91/16024; WO91/17424; U.S. Pat. No. 4,897,355; Bergan et al. 1993. Nucleic AcidsResearch. 21:3567). Also polyamine or polycation conjugates usingcompounds such as polylysine, protamine, or N1, N12-bis (ethyl) spermine(see, e.g., Bartzatt, R. et al. 1989. Biotechnol. Appl. Biochem. 11:133;Wagner E. et al. 1992. Proc. Natl. Acad. Sci. 88:4255) can be employed.In the case of a cell culture or tissue explant, the cells areconveniently incubated in a solution containing the chimeric RNA of theinvention (or a RNA agent) or lipid-mediated transfection; in the caseof a whole animal or plant, the chimeric RNA of the invention (or a RNAagent) is conveniently introduced by injection or perfusion into acavity or interstitial space of an organism, or systemically via oral,topical, parenteral (including subcutaneous, intramuscular andintravenous administration), vaginal, rectal, intranasal, ophthalmic, orintraperitoneal administration.

In addition, the chimeric RNA of the invention (or a RNA agent) can beadministered via an implantable extended release device. Methods fororal introduction include direct mixing of RNA with food of theorganism, as well as engineered approaches in which a species that isused as food is engineered to express an RNA, then fed to the organismto be affected. The chimeric RNA of the invention (or a RNA agent) maybe sprayed onto a plant or a plant may be genetically engineered toexpress the RNA in an amount sufficient to kill some or all of apathogen known to infect the plant.

1.4.2 Indirect Introduction of RNA

Alternatively, the RNA agent can be supplied to a cell indirectly byintroducing (e.g., by transformation or transfection) one or moreexpression constructs or expression vectors that encode the chimeric RNAmolecule of the invention. The expression of the chimeric RNA of theinvention can be transient or—for example after integration into thegenome (for example using selection markers) of the organism—stable.Preferably for pharmaceutical application, the RA agent is introducedtransiently, and not stably integrated into the genome. Preferably forapplications in plants, the chimeric RNA expression system is integratedstably into the genome—for example the chromosomal DNA or the DNA of theorganelles (for example the plastids (e.g., chloroplasts), mitochondriaand the like)—of a cell. Integration into the chromosomal DNA ispreferred.

Expression constructs and vectors are generally described above (seeDEFINITION section and section 1.3.1). Preferred expression constructsare described in more detailed below for the specific applications thecomposition and methods of the present invention. Methods for supplyinga cell with RNA by introducing an expression construct or vector fromwhich it can be transcribed are set forth in WO 99/32619. Principallyalso all the methods for direct introduction of RNA molecules into cellsas described above can be employed for introduction of the nucleic acidmolecules resembling the expression construct or vector.

2. Applications of Chimeric RNA of the Invention

The invention has broad opportunities of application, preferably in thefield of plants, human and animals. Generally, the methods and subjectmatter of the invention can be used to increase or decrease with higherspecificity the expression of any gene or sequence of interest includingtherapeutic or immunogenic peptides and proteins, nucleic acids forcontrolling gene expression, genes to reproduce enzymatic pathways forchemical synthesis, genes to shunt an enzymatic pathway for enhancedexpression of a particular intermediate or final product, industrialprocesses, and the like.

In one preferred embodiment, the eukaryotic organism is a plant and thepromoter is a promoter functional in plants. For plants, the expressednucleotide sequence preferably modulates expression of a gene involvedin agronomic traits, disease resistance, herbicide resistance, and/orgrain characteristics. The person skilled in art is aware of numerousnucleotide sequences which can be used in the context and for which aenhanced expression specificity is advantageous. The target nucleotidesequence comprises any nucleotide sequence or gene of interest,including genes, regulatory sequences, etc. Genes of interest includethose encoding agronomic traits, insect resistance, disease resistance,herbicide resistance, sterility, grain characteristics, and the like.The genes may be involved in metabolism of oil, starch, carbohydrates,nutrients, etc. Genes or traits of interest include, but are not limitedto, environmental- or stress-related traits, disease-related traits, andtraits affecting agronomic performance. Target sequences also includegenes responsible for the synthesis and/or degradation of proteins,peptides, fatty acids, lipids, waxes, oils, starches, sugars,carbohydrates, flavors, odors, toxins, carotenoids, hormones, polymers,flavonoids, storage proteins, phenolic acids, alkaloids, lignins,tannins, celluloses, glycoproteins, glycolipids, etc.

Various applications in plants are contemplated herein for whichmodulation of the expression profile in certain directions isadvantageous. This modulation is achieved by selection the microRNA-tagin a way, that the expression profile of the naturally occurring miRNAfits with the tissues, times, and/or under environmental conditionswhere no or lower expression should be achieved. For example, themicroRNA has a natural expression profile in the plant selected from thegroup consisting of

-   a) substantially constitutive expression but no expression in seed,-   b) predominant expression in seeds but not in other tissues,-   c) drought or other abiotic stress—induced expression,-   d) plant pathogen—induced expression,-   e) temporal expression (e.g., during early development, germination,    pollination etc.), and-   f) chemical induced expression.

Preferably, the microRNA is a plant microRNA selected from the groupconsisting of

-   a) the sequences as described by SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8,    9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,    26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,    43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 225, 226, 227,    228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240,    241, 242, 243, 245, 245, 246, 247, 248, 249, 250, 251, 252, 253,    254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, and 266,    and-   b) derivatives of the sequences described by SEQ ID NO: 1, 2, 3, 4,    5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,    23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,    40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 225,    226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238,    239, 240, 241, 242, 243, 245, 245, 246, 247, 248, 249, 250, 251,    252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264,    265, and 266.

A derivative is preferably a sequence, which fulfills the samefunctional endogenous purpose (e.g., certain gene control functions) inan organism of a different species (i.e. different from the specie wherethe disclosed miRNA is derived from). Said derivates may have certainmismatches with respect to the specifically disclosed sequences,preferably a derivative is characterized by having an identity of atleast 70%, preferably at least 80% or 85%, more preferably at least 90%,most preferably at least 95% to a sequence described by any of SEQ IDNO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238,239, 240, 241, 242, 243, 245, 245, 246, 247, 248, 249, 250, 251, 252,253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, and266. The mismatches with the specified miRNA maybe throughout the entiresequence but are preferably in the 3′ region of the miRNA (correspondingto the 5′-region of the complementary miRNA-tag). More specificapplications in plants are described herein below.

Other applications of the invention provide herein are used in animals(especially mammals) or human. Especially preferred are pharmaceuticalapplications. Thus, in another preferred embodiment of the invention thetarget organism is a mammal (more preferably a human being) and thepromoter is a promoter functional in mammals (more preferably inhumans). The expressed nucleotide sequence comprising the miRNA-tagpreferably modulates (e.g., express, over-express, or suppress)expression of a gene selected from the group consisting of genesinvolved in a human or animal disease or is a therapeutic gene.Alternatively, exogenous genes or sequences may be expressed which havea curative effect on the target organism. The disease is preferablyselected from the group of immunological diseases, cancer, diabetes,neurodegeneration, and metabolism diseases. The person skilled in theart is aware of numerous sequences which can be used in this context.The modulated gene may be selected from the group consisting ofretinoblastoma protein, p53, angiostatin, leptin, hormones, growthfactors, cytokines, insulin, growth hormones, alpha-interferon,beta-glucocerebrosidase, serum albumin, hemoglobin, and collagen.Therapeutic genes may be selected from the group consisting of tumornecrosis factor alpha (ADD). In this context the invention disclosedherein is a improved method for gene therapy or nucleotide-mediatedtherapy.

Various promoters are currently used in the art to express sequences inanimal, mammalian or human organism. Most of them are lackingtissue-specificity and can be advantageously combined with the teachingprovided herein. For example the promoter may be selected from groupconsisting of the perbB2 promoter, whey acidic protein promoter,stromelysin 3 promoter, prostate specific antigen promoter, probasinpromoter.

Various applications in animal, mammalian or human organisms arecontemplated herein for which modulation of the expression profile incertain directions is advantageous. This modulation is achieved byselection the microRNA-tag in a way, that the expression profile of thenaturally occurring miRNA fits with the tissues, times, and/or underenvironmental conditions where no or lower expression should beachieved. For example, the microRNA has a natural expression profile inthe animal, mammalian or human organism selected from the groupconsisting of

-   a) tissue specific expression in a tissue selected from the group    consisting of brain tissue, liver tissue, muscle tissue, neuron    tissue, and tumor tissue.-   b) stress-induced expression,-   c) pathogen-induced expression,-   d) neoplastic growth or tumorgenic growth induced expression, and-   e) age-dependent expression.

Preferably, the microRNA is an animal, mammalian or human microRNA.Hundreds of miRNAs have been cloned from mouse and human organs and celllines, and numerous additional miRNAs have been predicted withcomputational algorithms (Lagos-Quintana M et al. Science 2001,294:853-858; Lagos-Quintana M et al. Curr Biol 2002, 12:735-739;Lagos-Quintana M et al. RNA 2003, 9:175-179; Lim L P et al. Science2003, 299:1540; Mourelatos Z et al. Genes Dev 2002, 16:720-728; Dostie Jet al RNA 2003, 9:180-186). Various of these microRNAs and theirexpression profile are described in the art (see for example Sempere L Fet al. Genome Biology 2004, 5:R13; electronically available online atgenomebiology.com/2004/5/3/R13); hereby incorporated by referenceentirely including the cited references therein). Sempere et al.characterized the expression of 119 miRNAs in adult organs from mouseand human using northern blot analysis. Of these, 30 miRNAs werespecifically expressed or greatly enriched in a particular organ (brain,lung, liver or skeletal muscle). A total of 19 brain-expressed miRNAs(including lin-4 and let-7 orthologs) were coordinately upregulated inboth human and mouse embryonal carcinoma cells during neuronaldifferentiation. Mouse and human miRNAs often demonstrate a highhomology (about 90%) and may be interchangeable (see FIG. 5 in Sempereet al. 2004).

A total of 17 of the expressed miRNAs were detected exclusively in aparticular mouse organ; these included: seven brain-specific miRNAs(miR-9, -124a, -124b, -135, -153, -183, -219), six lung-specific miRNAs(miR-18, -19a, -24, -32, -130, -213), two spleen-specific miRNAs(miR-189, -212), one liverspecific miRNA (miR-122a), and oneheart-specific miRNA (miR-208) (Sempere et al. 2004; FIG. 2). Most ofthe indicated mouse brain-, liver and heart-specific miRNAs were alsodetected in the human counterpart organs. Among the 75 miRNAs that weredetected in two or more mouse organs, the levels of 14 of these weredetected in a particular mouse organ at levels at least two-fold higherthan in any other organ; these included: seven brain-enriched miRNAs(miR-9*, -125a, -125b, -128, -132, -137, -139), three skeletalmuscle-enriched miRNAs (miR-1d, -133, -206), two kidney-enriched miRNAs(miR-30b, -30c), and one spleen-enriched miRNA (miR-99a). Allbrain-enriched and skeletal muscle-enriched miRNAs had similar elevatedlevels in the human counterpart organs. There is a high conservation ofexpression of these organ-specific and organ-enriched miRNAs betweenmouse and human.

A group of six miRNAs was expressed primarily in mouse spleen (miR-127,-142-a, -142-s, -151, -189, -212). A group of five miRNAs was expressedin mouse and human liver (miR-122a, -152, -194, -199, -215) with somescattered expression in other organs including lung and kidney.

A group of seven miRNAs was expressed in mouse lung and kidney (miR-18,-20, -24, -32, -141, -193, -200b). Together, the last two groups mightreflect a role of miRNAs in an epithelial cell type since liver, lung,and kidney are organs containing epithelial tissues.

A group of 17 miRNAs was expressed in mouse and human brain (miR-7, -9,-9*, -124a, -124b, -125a, -125b, -128, -132, -135, -137, -139, -153,-149, -183, -190, -219) with scattered expression in other organs.

A group of six miRNAs was expressed in mouse and human skeletal muscleand heart: miR-1b, -1d, -133 and -206 had elevated expression in heartand skeletal muscle with low expression in other organs and miR-143 and-208 were almost exclusively detected in heart and skeletal muscle.

A group of five miRNAs showed abundant expression across organs (let-7a,-7b, miR-30b, -30c).

The miRNA sequences are well known in the art and described for examplein Sempere et al. 2004 and the additional electronically available datafor this paper. For example some of the miRNA-tags are specified herein:hsa-miR-19a (SEQ ID NO: 90), hsa-let7b (SEQ ID NO: 91), hsa-miR-100 (SEQID NO: 92), hsa-miR-103-1 (SEQ ID NO: 93), hsa-miR-107 (SEQ ID NO: 94),hsa-miR-10a (SEQ ID NO: 95), hsa-miR-124b (SEQ ID NO: 96), hsa-miR-129a(SEQ ID NO: 97), hsa-miR-139 (SEQ ID NO: 98), hsa-miR-147 (SEQ ID NO:99), hsa-miR-148 (SEQ ID NO: 100), hsa-miR-15a (SEQ ID NO: 101),hsa-miR-16 (SEQ ID NO: 102), hsa-miR-18 (SEQ ID NO: 103), hsa-miR-192(SEQ ID NO: 104), hsa-miR-196 (SEQ ID NO: 105), hsa-miR-199a (SEQ ID NO:106), hsa-let7a (SEQ ID NO: 107), hsa-miR-24 (SEQ ID NO: 108),hsa-miR-20 (SEQ ID NO: 109), hsa-miR-208 (SEQ ID NO: 110), hsa-miR-210(SEQ ID NO: 111), hsa-miR-212 (SEQ ID NO: 112), hsa-miR-213 (SEQ ID NO:113), hsa-miR-214 (SEQ ID NO: 114), hsa-miR-215 (SEQ ID NO: 115),hsa-miR-216 (SEQ ID NO: 116), hsa-miR-217 (SEQ ID NO: 117), hsa-miR-218(SEQ ID NO: 118), hsa-miR-219 (SEQ ID NO: 119), hsa-miR-22 (SEQ ID NO:120), hsa-miR-220 (SEQ ID NO: 121), hsa-miR-221 (SEQ ID NO: 122),hsa-miR-222 (SEQ ID NO: 123), hsa-miR-23a (SEQ ID NO:124), hsa-miR-19b(SEQ ID NO: 125), hsa-miR-96 (SEQ ID NO: 126), hsa-miR-26b (SEQ ID NO:127), hsa-miR-27a (SEQ ID NO: 128), hsa-miR-28 (SEQ ID NO: 129),hsa-miR-29 (SEQ ID NO: 130), hsa-miR-29b (SEQ ID NO: 131), hsa-miR-30a(SEQ ID NO: 132), hsa-miR-30c (SEQ ID NO: 133), hsa-miR-30d (SEQ ID NO:134), hsa-miR-30e (SEQ ID NO: 135), hsa-miR-32 (SEQ ID NO: 136),hsa-miR-33 (SEQ ID NO: 137), hsa-miR-7 (SEQ ID NO: 138), hsa-miR-91 (SEQID NO: 139), hsa-miR-92 (SEQ ID NO: 140), hsa-miR-93 (SEQ ID NO: 141),hsa-miR-95 (SEQ ID NO: 142), hsa-miR-98 (SEQ ID NO: 143), hsa-miR-26a(SEQ ID NO: 144). These sequences specify the potential miRNA-tag. Thecorresponding miRNA is the complementary sequence in RNA.

Preferably, the microRNA is an animal, mammalian or human microRNAselected from the group consisting of

-   a) the sequences as described by SEQ ID NO: 56, 57, 58, 59, 60, 61,    62, and 63, and-   b) derivatives of the sequences described by SEQ ID NO: 56, 57, 58,    59, 60, 61, 62, and 63, and-   c) the complementary RNA sequence to a sequence as described by any    of SEQ ID NO: 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102,    103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,    116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,    129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,    142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,    155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167,    168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180,    181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193,    194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206,    207, or 208, and-   d) derivatives of RNA sequence complementary to a sequence as    described by any of SEQ ID NO: 90, 91, 92, 93, 94, 95, 96, 97, 98,    99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,    113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,    126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138,    139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151,    152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164,    165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177,    178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190,    191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203,    204, 205, 206, 207, or 208.

A derivative is preferably a sequence, which fulfills the samefunctional endogenous purpose (e.g., certain gene control functions) inan organism of a different species (i.e. different from the specie wherethe disclosed miRNA is derived from). Said derivates may have certainmismatches with respect to the specifically disclosed sequences,preferably a derivative is characterized by having an identity of atleast 70%, preferably at least 80% or 85%, more preferably at least 90%,most preferably at least 95% to a sequence described by any of SEQ IDNO: 56, 57, 58, 59, 60, 61, 62, and 63 or a RNA sequence complementaryto a sequence as described by any of SEQ ID NO: 90, 91, 92, 93, 94, 95,96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110,111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124,125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138,139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152,153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166,167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180,181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194,195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, or 208.The mismatches with the specified miRNA maybe throughout the entiresequence but are preferably in the 3′ region (corresponding to the5′-region of the miRNA tag).

Another embodiment of the invention relates to a pharmaceuticallypreparation of at least one expression construct, a chimericribonucleotide sequence, or a vector according to the invention.

More specific applications in animals and humans, especially in thefield of pharmaceutical applications are described herein below.

As mentioned above the method and subject matter of the invention can beemployed to increase specificity of expression for chimeric nucleotidesequence, which may encode

-   i) a protein (i.e. by comprising an open reading frame (ORF),-   ii) a functional RNA (e.g., a antisense, sense, double-stranded or    ribozyme RNA), which is preferably employed in a gene silencing    approach.

2.1 Expression with Enhanced Specificity

The method for expression or over-expression of nucleotide sequences invarious organism is well known to the person skilled in the art(Maniatis T, Fritsch E F and Sambrook J (1989) Molecular Cloning: ALaboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold SpringHarbor (N.Y.); Silhavy et al. (1984) Experiments with Gene Fusions, ColdSpring Harbor Laboratory, Cold Spring Harbor (N.Y.); Ausubel et al.(1987) Current Protocols in Molecular Biology, Greene Publishing Assoc.and Wiley Interscience; see section 3.3 for some details).

2.2 Gene Silencing with Enhanced Specificity

The chimeric RNA molecules of the invention, the expression constructsand the expression vectors for their expression, and the transgenicorganism comprising said molecules could be utilized in gene silencing(i.e. to attenuate, reduce or suppress expression of target genes intarget cells or organism). For this the chimeric RNA may comprisesequences, which are capable to provide an antisense RNA (for antisenseRNA mediated gene silencing), double-stranded RNA (for dsRNAinterference gene silencing) or senseRNA (for co-suppression genesilencing)

The methods of the invention will lead to better results and/or higherefficiencies when compared to the methods using conventional sense,antisense, or double-stranded RNA nucleotide sequences.

Another embodiment of the invention relates to composition for altering,preferably reducing or attenuating, expression of a target gene,comprising at least one chimeric RNA of the invention. Yet anotherembodiment of the invention relates to a method for attenuating (orreducing or suppressing) expression of at least one target gene in aneukaryotic cell, comprising introducing a chimeric RNA molecule of theinvention (or an expression construct or vector encoding the same) intothe cells in an amount sufficient to attenuate expression of the targetgene, wherein the chimeric RNA molecule comprises at least oneribonucleotide sequence that is substantially identical to at least apart of the nucleotide sequence of the target gene.

Any gene being expressed in a cell (preferably an eukaryotic cell) canbe targeted. A gene that is expressed in the cell is one that istranscribed to yield a RNA (e.g., a mRNA) and, optionally, a protein.Preferably the target gene is a eukaryotic gene, more preferably amammalian, nematode, fungal or plant gene. Preferably the target gene isan endogenous gene of the cell or a heterologous gene relative to thegenome of the cell, such as a pathogen gene. Preferably, the gene of apathogen is from a pathogen capable to infect an eukaryotic organism.Most preferably, said pathogen is selected from the group of virus,bacteria, fungi and nematodes.

The chimeric RNA may be produced outside the cell (i.e. the host cell inwhich gene silencing should be achieved), or may be recombinantlyproduced by an expression construct or expression vector within thecell. The host cell is preferably eukaryotic cell, more preferably anematode, mammalian cell or a plant cell.

Preferably, the target gene expression is attenuated (or reduced orsuppressed) by at least about 10%, preferably at least about 30%, morepreferably at least about 50%, even more preferably at least about 70%,most preferably at least about 90%.

To achieve gene silencing the chimeric RNA of the invention comprises atleast one ribonucleotide sequence that is substantially identical (asdefined above), preferably identical, to at least a part of at least onetarget gene. Preferably, said part of a target gene having substantialidentity to said ribonucleotide sequence has a length of least 15nucleotides, preferably at least 19 nucleotides, more preferably atleast 50 nucleotides, even more preferably at least 100 nucleotides,most preferably at least 250 nucleotides. More preferably, saidnucleotide sequence has an identity of at least 65%, preferably at least80%, more preferably at least 90%, most preferably 95%, even morepreferably 100% to a sequence of at least 15 nucleotides, preferably atleast 19 nucleotides, more preferably at least 50 nucleotides, even morepreferably 100 nucleotides, most preferably at least 250 nucleotides ofat least one target gene. Preferably, said first ribonucleotide sequencehybridizes (preferably under stringent conditions, more preferably underlow stringency conditions, most preferably under high stringencyconditions) to a sequence of the target gene.

In a preferred embodiment the nucleotide sequence is substantiallyidentical, preferably identical, to a part of the coding sequence or thenon-coding sequence of the target gene (preferably an eukaryotic gene,such as a mammalian or plant gene). The non-coding sequence can be the5′- or 3′-untranslated sequence or the introns but can also be anontranscribed sequence. Non-coding sequences as target sequence arepreferred in cases where the target gene encodes a member of a genefamily (i.e. different genes encoding very similar proteins).

The target gene can be an endogenous gene or an exogenous or foreigngene (i.e., a transgene or a pathogen gene). For example, a transgenethat is present in the genome of a cell as a result of genomicintegration of the viral delivery construct can be regulated usingchimeric RNA according to the invention. The foreign gene can beintegrated into the host genome (preferably the chromosomal DNA), or itmay be present on an extrachromosomal genetic construct such as aplasmid or a cosmid. For example, the target gene may be present in thegenome of the cell into which the chimeric RNA is introduced, or in thegenome of a pathogen, such as a virus, a bacterium, a fungus or aprotozoan, which is capable of infecting such organism or cell.

The eukaryotic cell or organism to which the chimeric RNA of theinvention can be delivered can be derived from any eukaryotic organism,such as for example without limitation, plants or animals, such asmammals, insects, nematodes, fungi, algae, fish, and birds. Likewise,the chimeric RNA molecule of the invention or the expression constructsor vectors for its expression can be used to suppress or reduce anytarget gene in any eukaryotic organism. In some embodiments of theinvention also prokaryotic organism comprising the chimeric RNA of theinvention are useful. For example prokaryotic cells and organism can beused to produce or amplify the chimeric RNA of the invention or anexpression construct or vector encoding the same. Furthermore,prokaryotic organism can be utilized as vehicles to introduce thechimeric RNA of the invention into animals e.g. by feeding. Also,prokaryotic organisms, for example Agrobacteria, can advantageously beemployed as vehicles for the transformation of, for example, plantorganisms.

Thus a further aspect of the invention relates to cells and organism(e.g., plant, animal, protozoan, virus, bacterium, or fungus), whichcomprise at least one chimeric RNA of the invention, or an expressionconstruct or expression vectors encoding said chimeric RNA molecule (asdefined above in more detail).

Another embodiment of the present invention relates to a method forattenuating (or reducing) expression of at least one target gene in aneukaryotic cell, comprising introducing a chimeric RNA molecule of theinvention into the cell in an amount sufficient to attenuate expressionof the target gene, wherein the chimeric RNA molecule comprises at leastone ribonucleotide sequence that is substantially identical, preferablyidentical, to at least part of the nucleotide sequence of the targetgene. Depending on the particular target gene and the dose of chimericRNA delivered, the method may partially or completely inhibit expressionof the gene in the cell. Preferably the chimeric RNA of the invention iscapable of effectively eliminating, substantially reducing, or at leastpartially reducing the level of a RNA (preferably mRNA) transcript orprotein encoded by the target gene (or gene family). Preferably, theexpression of the target gene (as measured by the expressed RNA orprotein) is reduced, inhibited or attenuated by at least 10%, preferablyat least 30% or 40%, preferably at least 50% or 60%, more preferably atleast 80%, most preferably at least 90% or 95%. The levels of targetproducts such as transcripts or proteins may be decreased throughout anorganism such as a plant or mammal, or such decrease in target productsmay be localized in one or more specific organs or tissues of theorganism. For example, the levels of products may be decreased in one ormore of the tissues and organs of a plant including without limitation:roots, tubers, stems, leaves, stalks, fruit, berries, nuts, bark, pods,seeds and flowers. A preferred organ is a seed of a plant.

The expression of two or more genes can be attenuated concurrently byintroducing two or more chimeric RNAs or one RNA capable to provide(e.g., by subsequent RNA processing) more than one chimeric RNA moleculeinto the cell in amounts sufficient to attenuate expression of theirrespective target genes.

To overcome the sequence-independent protein kinase PKR stress-responsetriggered by dsRNA, modifications are made to a chimeric RNA molecule,which would normally activate the interferon pathway such that theinterferon pathway is not activated. In certain embodiments, the cellscan be treated with an agent(s) that inhibits the general dsRNAresponse(s) by the host cells, such as may give rise tosequence-independent apoptosis. For instance, the cells can be treatedwith agents that inhibit the dsRNA-dependent protein kinase known as PKR(protein kinase RNA-activated). Likewise, overexpression of agents,which ectopically activate eIF2α□ can be used. Other agents, which canbe used to suppress the PKR response, include inhibitors of IκBphosphorylation of IκB, inhibitors of IκB ubiquitination, inhibitors ofI□B degradation, inhibitors of NFκB nuclear translocation, andinhibitors of NF-□B interaction with κB response elements. Otherinhibitors of sequence-independent dsRNA response in cells include thegene product of the vaccinia virus E3L. The E3L gene product containstwo distinct domains. A conserved carboxyterminal domain has been shownto bind dsRNA and inhibit the antiviral dsRNA response by cells.Expression of at least that portion of the E3L gene in the host cell, orthe use of polypeptide or peptidomimetics thereof, can be used tosuppress the general dsRNA response. Caspase inhibitors sensitize cellsto killing by dsRNA. Accordingly, ectopic expression or activation ofcaspases in the host cell can be used to suppress the general dsRNAresponse.

3. Specific Applications

The subsequent application of compositions and methods according to theinvention may be mentioned by way of example, but not by limitation:

3.1 Applications in Plant Biotechnology

The method according to the invention is preferably employed for thepurposes of plant biotechnology for generating plants with advantageousproperties. Thus, the suitability of the plants or their seeds asfoodstuff or feeding stuff can be improved, for example via amodification of the compositions and/or the content of metabolites, inparticular proteins, oils, vitamins and/or starch. Also, growth rate,yield or resistance to biotic or abiotic stress factors can beincreased. The subsequent applications in the field of plantbiotechnology are particularly advantageous.

A further aspect of the invention relates to a transgenic plant or plantcell comprising a chimeric RNA of the invention, or an expressionconstruct or expression vector for expression of said chimeric RNA.Another embodiment relates to the use of the transgenic organismaccording to the invention (e.g., the transgenic plant) and of thecells, cell cultures, parts—such as, for example, in the case oftransgenic plant organisms roots, leaves and the like—derived from themand transgenic propagation material such as seeds or fruits for theproduction of foodstuffs or feeding stuffs, pharmaceuticals or finechemicals, such as, for example, enzymes, vitamins, amino acids, sugars,fatty acids, natural or synthetic flavorings, aromas and colorants.Especially preferred is the production of triacylglycerides, lipids,oils, fatty acids, starches, tocopherols and tocotrienols andcarotenoids. Genetically modified plants according to the inventionwhich can be consumed by humans and animals can also be used asfoodstuffs or feeding stuffs, for example directly or after undergoing aprocessing which is known per se.

3.1.1 Plant Target Genes for Expression with Enhanced Specificity

3.1.1.1. Herbicide Resistance

The genes encoding phosphinothricin acetyltransferase (bar and pat),glyphosate tolerant EPSP synthase genes, the glyphosate degradativeenzyme gene gox encoding glyphosate oxidoreductase, deh (encoding adehalogenase enzyme that inactivates dalapon), herbicide resistant(e.g., sulfonylurea and imidazolinone) acetolactate synthase, and bxngenes (encoding a nitrilase enzyme that degrades bromoxynil) are goodexamples of herbicide resistant genes for use in transformation. The barand pat genes code for an enzyme, phosphinothricin acetyltransferase(PAT), which inactivates the herbicide phosphinothricin and preventsthis compound from inhibiting glutamine synthetase enzymes. The enzyme5-enolpyruvylshikimate 3-phosphate synthase (EPSP Synthase), is normallyinhibited by the herbicide N-(phosphonomethyl)glycine (glyphosate).However, genes are known that encode glyphosate-resistant EPSP Synthaseenzymes. The deh gene encodes the enzyme dalapon dehalogenase andconfers resistance to the herbicide dalapon. The bxn gene codes for aspecific nitrilase enzyme that converts bromoxynil to a non-herbicidaldegradation product.

For this application either a miRNA-tag, which allows for enhancedspecific expression in green leafs is preferred for designing themiRNA-tag. For example, Arabidopsis miR160b expressed in root andflower, but not in the leafs is good for such application.

3.1.1.2 Insect Resistance

An important aspect of the present invention concerns the introductionof insect resistance-conferring genes into plants. Potential insectresistance genes which can be introduced include Bacillus thuringiensiscrystal toxin genes or Bt genes (Watrud 1985). Bt genes may provideresistance to lepidopteran or coleopteran pests such as European CornBorer (ECB) and corn rootworm (CRW). Preferred Bt toxin genes for use insuch embodiments include the CryIA(b) and CryIA(c) genes. Endotoxingenes from other species of B. thuringiensis which affect insect growthor development may also be employed in this regard. Protease inhibitorsmay also provide insect resistance (Johnson 1989), and will thus haveutility in plant transformation. The use of a protease inhibitor IIgene, pinII, from tomato or potato is envisioned to be particularlyuseful. Even more advantageous is the use of a pinII gene in combinationwith a Bt toxin gene, the combined effect of which has been discoveredby the present inventors to produce synergistic insecticidal activity.Other genes which encode inhibitors of the insects' digestive system, orthose that encode enzymes or co-factors that facilitate the productionof inhibitors, may also be useful. This group may be exemplified bycystatin and amylase inhibitors, such as those from wheat and barley.

Also, genes encoding lectins may confer additional or alternativeinsecticide properties. Lectins (originally termed phytohemagglutinins)are multivalent carbohydrate-binding proteins which have the ability toagglutinate red blood cells from a range of species. Lectins have beenidentified recently as insecticidal agents with activity againstweevils, ECB and rootworm (Murdock 1990; Czapla & Lang, 1990). Lectingenes contemplated to be useful include, for example, barley and wheatgerm agglutinin (WGA) and rice lectins (Gatehouse 1984), with WGA beingpreferred.

Genes controlling the production of large or small polypeptides activeagainst insects when introduced into the insect pests, such as, e.g.,lytic peptides, peptide hormones and toxins and venoms, form anotheraspect of the invention. For example, it is contemplated, that theexpression of juvenile hormone esterase, directed towards specificinsect pests, may also result in insecticidal activity, or perhaps causecessation of metamorphosis (Hammock 1990).

Transgenic plants expressing genes which encode enzymes that affect theintegrity of the insect cuticle form yet another aspect of theinvention. Such genes include those encoding, e.g., chitinase,proteases, lipases and also genes for the production of nikkomycin, acompound that inhibits chitin synthesis, the introduction of any ofwhich is contemplated to produce insect resistant maize plants. Genesthat code for activities that affect insect molting, such thoseaffecting the production of ecdysteroid UDP-glucosyl transferase, alsofall within the scope of the useful transgenes of the present invention.

Genes that code for enzymes that facilitate the production of compoundsthat reduce the nutritional quality of the host plant to insect pestsare also encompassed by the present invention. It may be possible, forinstance, to confer insecticidal activity on a plant by altering itssterol composition. Sterols are obtained by insects from their diet andare used for hormone synthesis and membrane stability. Thereforealterations in plant sterol composition by expression of novel genes,e.g., those that directly promote the production of undesirable sterolsor those that convert desirable sterols into undesirable forms, couldhave a negative effect on insect growth and/or development and henceendow the plant with insecticidal activity. Lipoxygenases are naturallyoccurring plant enzymes that have been shown to exhibit anti-nutritionaleffects on insects and to reduce the nutritional quality of their diet.Therefore, further embodiments of the invention concern transgenicplants with enhanced lipoxygenase activity which may be resistant toinsect feeding.

The present invention also provides methods and compositions by which toachieve qualitative or quantitative changes in plant secondarymetabolites. One example concerns transforming plants to produce DIMBOAwhich, it is contemplated, will confer resistance to European cornborer, rootworm and several other maize insect pests. Candidate genesthat are particularly considered for use in this regard include thosegenes at the bx locus known to be involved in the synthetic DIMBOApathway (Dunn 1981). The introduction of genes that can regulate theproduction of maysin, and genes involved in the production of dhurrin insorghum, is also contemplated to be of use in facilitating resistance toearworm and rootworm, respectively.

Tripsacum dactyloides is a species of grass that is resistant to certaininsects, including corn root worm. It is anticipated that genes encodingproteins that are toxic to insects or are involved in the biosynthesisof compounds toxic to insects will be isolated from Tripsacum and thatthese novel genes will be useful in conferring resistance to insects. Itis known that the basis of insect resistance in Tripsacum is genetic,because said resistance has been transferred to Zea mays via sexualcrosses (Branson & Guss, 1972).

Further genes encoding proteins characterized as having potentialinsecticidal activity may also be used as transgenes in accordanceherewith. Such genes include, for example, the cowpea trypsin inhibitor(CpTI; Hilder 1987) which may be used as a rootworm deterrent; genesencoding avermectin (Campbell 1989; Ikeda 1987) which may proveparticularly useful as a corn rootworm deterrent; ribosome inactivatingprotein genes; and even genes that regulate plant structures. Transgenicmaize including anti-insect antibody genes and genes that code forenzymes that can covert a non-toxic insecticide (proinsecticide) appliedto the outside of the plant into an insecticide inside the plant arealso contemplated.

For this application either a miRNA-tag, which allows for enhancedspecific expression in tissue, which presents the interaction or entryside for the insect (or other pathogen) (e.g., the epidermis) or amiRNA-tag corresponding to an miRNA, which is endogenously suppressed bythe insect or pathogen induced stress factor is preferred to be employedfor designing the miRNA-tag. For example, maize miR167 is predominantlyexpressed in seed, use of Zm miR167 tag in a transgene constructexpressing insecticidal molecules can prevent leaky expression of suchmolecules in the seeds. Some of them (e.g. lectin) is a potentialallergen for human.

3.1.1.3 Environment or Stress Resistance

Improvement of a plant's ability to tolerate various environmentalstresses such as, but not limited to, drought, excess moisture,chilling, freezing, high temperature, salt, and oxidative stress, canalso be effected through expression of heterologous, or overexpressionof homologous genes. Benefits may be realized in terms of increasedresistance to freezing temperatures through the introduction of an“antifreeze” protein such as that of the Winter Flounder (Cutler 1989)or synthetic gene derivatives thereof. Improved chilling tolerance mayalso be conferred through increased expression of glycerol-3-phosphateacetyltransferase in chloroplasts (Murata 1992; Wolter 1992). Resistanceto oxidative stress (often exacerbated by conditions such as chillingtemperatures in combination with high light intensities) can beconferred by expression of superoxide dismutase (Gupta 1993), and may beimproved by glutathione reductase (Bowler 1992). Such strategies mayallow for tolerance to freezing in newly emerged fields as well asextending later maturity higher yielding varieties to earlier relativematurity zones.

Expression of novel genes that favorably effect plant water content,total water potential, osmotic potential, and turgor can enhance theability of the plant to tolerate drought. As used herein, the terms“drought resistance” and “drought tolerance” are used to refer to aplants increased resistance or tolerance to stress induced by areduction in water availability, as compared to normal circumstances,and the ability of the plant to function and survive in lower-waterenvironments, and perform in a relatively superior manner. In thisaspect of the invention it is proposed, for example, that the expressionof a gene encoding the biosynthesis of osmotically-active solutes canimpart protection against drought. Within this class of genes are DNAsencoding mannitol dehydrogenase (Lee and Saier, 1982) andtrehalose-6-phosphate synthase (Kaasen 1992). Through the subsequentaction of native phosphatases in the cell or by the introduction andcoexpression of a specific phosphatase, these introduced genes willresult in the accumulation of either mannitol or trehalose,respectively, both of which have been well documented as protectivecompounds able to mitigate the effects of stress. Mannitol accumulationin transgenic tobacco has been verified and preliminary results indicatethat plants expressing high levels of this metabolite are able totolerate an applied osmotic stress (Tarczynski 1992).

Similarly, the efficacy of other metabolites in protecting either enzymefunction (e.g. alanopine or propionic acid) or membrane integrity (e.g.,alanopine) has been documented (Loomis 1989), and therefore expressionof gene encoding the biosynthesis of these compounds can confer droughtresistance in a manner similar to or complimentary to mannitol. Otherexamples of naturally occurring metabolites that are osmotically activeand/or provide some direct protective effect during drought and/ordesiccation include sugars and sugar derivatives such as fructose,erythritol (Coxson 1992), sorbitol, dulcitol (Karsten 1992),glucosylglycerol (Reed 1984; Erdmann 1992), sucrose, stachyose (Koster &Leopold 1988; Blackman 1992), ononitol and pinitol (Vernon & Bohnert1992), and raffinose (Bernal-Lugo & Leopold 1992). Other osmoticallyactive solutes which are not sugars include, but are not limited to,proline and glycine-betaine (Wyn-Jones and Storey, 1981). Continuedcanopy growth and increased reproductive fitness during times of stresscan be augmented by introduction and expression of genes such as thosecontrolling the osmotically active compounds discussed above and othersuch compounds, as represented in one exemplary embodiment by the enzymemyoinositol 0-methyltransferase.

It is contemplated that the expression of specific proteins may alsoincrease drought tolerance. Three classes of Late Embryogenic Proteinshave been assigned based on structural similarities (see Dure 1989). Allthree classes of these proteins have been demonstrated in maturing(i.e., desiccating) seeds. Within these 3 types of proteins, the Type-11(dehydrin-type) have generally been implicated in drought and/ordesiccation tolerance in vegetative plant parts (e.g. Mundy and Chua,1988; Piatkowski 1990; Yamaguchi-Shinozaki 1992). Recently, expressionof a Type-III LEA (HVA-1) in tobacco was found to influence plantheight, maturity and drought tolerance (Fitzpatrick, 1993). Expressionof structural genes from all three groups may therefore confer droughttolerance. Other types of proteins induced during water stress includethiol proteases, aldolases and transmembrane transporters (Guerrero1990), which may confer various protective and/or repair-type functionsduring drought stress. The expression of a gene that effects lipidbiosynthesis and hence membrane composition can also be useful inconferring drought resistance on the plant.

Many genes that improve drought resistance have complementary modes ofaction. Thus, combinations of these genes might have additive and/orsynergistic effects in improving drought resistance in maize. Many ofthese genes also improve freezing tolerance (or resistance); thephysical stresses incurred during freezing and drought are similar innature and may be mitigated in similar fashion. Benefit may be conferredvia constitutive expression of these genes, but the preferred means ofexpressing these novel genes may be through the use of a turgor-inducedpromoter (such as the promoters for the turgor-induced genes describedin Guerrero et al. 1990 and Shagan 1993). Spatial and temporalexpression patterns of these genes may enable maize to better withstandstress.

Expression of genes that are involved with specific morphological traitsthat allow for increased water extractions from drying soil would be ofbenefit. For example, introduction and expression of genes that alterroot characteristics may enhance water uptake. Expression of genes thatenhance reproductive fitness during times of stress would be ofsignificant value. For example, expression of DNAs that improve thesynchrony of pollen shed and receptiveness of the female flower parts,i.e., silks, would be of benefit. In addition, expression of genes thatminimize kernel abortion during times of stress would increase theamount of grain to be harvested and hence be of value. Regulation ofcytokinin levels in monocots, such as maize, by introduction andexpression of an isopentenyl transferase gene with appropriateregulatory sequences can improve monocot stress resistance and yield(Gan 1995).

Given the overall role of water in determining yield, it is contemplatedthat enabling plants to utilize water more efficiently, through theintroduction and expression of novel genes, will improve overallperformance even when soil water availability is not limiting. Byintroducing genes that improve the ability of plants to maximize waterusage across a full range of stresses relating to water availability,yield stability or consistency of yield performance may be realized.

Improved protection of the plant to abiotic stress factors such asdrought, heat or chill, can also be achieved—for example—byoverexpressing antifreeze polypeptides from Myoxocephalus Scorpius (WO00/00512), Myoxocephalus octodecemspinosus, the Arabidopsis thalianatranscription activator CBF1, glutamate dehydrogenases (WO 97/12983, WO98/11240), calcium-dependent protein kinase genes (WO 98/26045),calcineurins (WO 99/05902), casein kinase from yeast (WO 02/052012),farnesyltransferases (WO 99/06580; Pei Z M et al. (1998) Science282:287-290), ferritin (Deak M et al. (1999) Nature Biotechnology17:192-196), oxalate oxidase (WO 99/04013; Dunwell J M (1998) BiotechnGenet Eng Rev 15:1-32), DREB1A factor (“dehydration response element B1A”; Kasuga M et al. (1999) Nature Biotech 17:276-286), genes ofmannitol or trehalose synthesis such as trehalose-phosphate synthase ortrehalose-phosphate phosphatase (WO 97/42326) or by inhibiting genessuch as trehalase (WO 97/50561).

For this application either a miRNA-tag, which allows for enhancedspecific expression in stress-sensitive tissue (e.g., young seedling orembryo) or a miRNA-tag corresponding to an miRNA, which is endogenouslysuppressed by the stress factor is preferred to be employed fordesigning the miRNA-tag.

3.1.1.4 Disease Resistance

It is proposed that increased resistance to diseases may be realizedthrough introduction of genes into plants period. It is possible toproduce resistance to diseases caused, by viruses, bacteria, fungi, rootpathogens, insects and nematodes. It is also contemplated that controlof mycotoxin producing organisms may be realized through expression ofintroduced genes.

Resistance to viruses may be produced through expression of novel genes.For example, it has been demonstrated that expression of a viral coatprotein in a transgenic plant can impart resistance to infection of theplant by that virus and perhaps other closely related viruses (Cuozzo1988, Hemenway 1988, Abel 1986). It is contemplated that expression ofantisense genes targeted at essential viral functions may impartresistance to said virus. For example, an antisense gene targeted at thegene responsible for replication of viral nucleic acid may inhibit saidreplication and lead to resistance to the virus. It is believed thatinterference with other viral functions through the use of antisensegenes may also increase resistance to viruses. Further it is proposedthat it may be possible to achieve resistance to viruses through otherapproaches, including, but not limited to the use of satellite viruses.

It is proposed that increased resistance to diseases caused by bacteriaand fungi may be realized through introduction of novel genes. It iscontemplated that genes encoding so-called “peptide antibiotics,”pathogenesis related (PR) proteins, toxin resistance, and proteinsaffecting host-pathogen interactions such as morphologicalcharacteristics will be useful. Peptide antibiotics are polypeptidesequences which are inhibitory to growth of bacteria and othermicroorganisms. For example, the classes of peptides referred to ascecropins and magainins inhibit growth of many species of bacteria andfungi. It is proposed that expression of PR proteins in plants may beuseful in conferring resistance to bacterial disease. These genes areinduced following pathogen attack on a host plant and have been dividedinto at least five classes of proteins (Bol 1990). Included amongst thePR proteins are β-1,3-glucanases, chitinases, and osmotin and otherproteins that are believed to function in plant resistance to diseaseorganisms. Other genes have been identified that have antifungalproperties, e.g., UDA (stinging nettle lectin) and hevein (Broakgert1989; Barkai-Golan 1978). It is known that certain plant diseases arecaused by the production of phytotoxins. Resistance to these diseasescould be achieved through expression of a novel gene that encodes anenzyme capable of degrading or otherwise inactivating the phytotoxin.Expression novel genes that alter the interactions between the hostplant and pathogen may be useful in reducing the ability the diseaseorganism to invade the tissues of the host plant, e.g., an increase inthe waxiness of the leaf cuticle or other morphological characteristics.

Plant parasitic nematodes are a cause of disease in many plants. It isproposed that it would be possible to make the plant resistant to theseorganisms through the expression of novel genes. It is anticipated thatcontrol of nematode infestations would be accomplished by altering theability of the nematode to recognize or attach to a host plant and/orenabling the plant to produce nematicidal compounds, including but notlimited to proteins.

Furthermore, a resistance to fungi, insects, nematodes and diseases, canbe achieved by by targeted accumulation of certain metabolites orproteins. Such proteins include but are not limited to glucosinolates(defense against herbivores), chitinases or glucanases and other enzymeswhich destroy the cell wall of parasites, ribosome-inactivating proteins(RIPs) and other proteins of the plant resistance and stress reaction asare induced when plants are wounded or attacked by microbes, orchemically, by, for example, salicylic acid, jasmonic acid or ethylene,or lysozymes from nonplant sources such as, for example, T4-lysozyme orlysozyme from a variety of mammals, insecticidal proteins such asBacillus thuringiensis endotoxin, a-amylase inhibitor or proteaseinhibitors (cowpea trypsin inhibitor), lectins such as wheatgermagglutinin, RNAses or ribozymes. Further examples are nucleic acidswhich encode the Trichoderma harzianum chit42 endochitinase (GenBankAcc. No.: S78423) or the N-hydroxylating, multi-functional cytochromeP-450 (CYP79) protein from Sorghum bicolor (GenBank Acc. No.: U32624),or functional equivalents of these. The accumulation of glucosinolatesas protection from pests (Rask L et al. (2000) Plant Mol Biol 42:93-113;Menard R et al. (1999) Phytochemistry 52:29-35), the expression ofBacillus thuringiensis endotoxins (Vaeck et al. (1987) Nature 328:33-37)or the protection against attack by fungi, by expression of chitinases,for example from beans (Broglie et al. (1991) Science 254:1194-1197), isadvantageous. Resistance to pests such as, for example, the rice pestNilaparvata lugens in rice plants can be achieved by expressing thesnowdrop (Galanthus nivalis) lectin agglutinin (Rao et al. (1998) PlantJ 15(4):469-77). The expression of synthetic cryIA(b) and cryIA(c)genes, which encode lepidoptera-specific Bacillus thuringiensisD-endotoxins can bring about a resistance to insect pests in variousplants (Goyal R K et al. (2000) Crop Protection 19(5):307-312). Furthertarget genes which are suitable for pathogen defense comprise“polygalacturonaseinhibiting protein” (PGIP), thaumatine, invertase andantimicrobial peptides such as lactoferrin (Lee T J et al. (2002) J AmerSoc Horticult Sci 127(2):158-164).

For this application either a miRNA-tag, which allows for enhancedspecific expression in tissue, which presents the interaction or entryside for the pathogen (e.g., the epidermis) or a miRNA-tag correspondingto an miRNA, which is endogenously suppressed by the pathogen inducedstress factor is preferred to be employed for designing the miRNA-tag.For example, maize miR167 is predominantly expressed in seeds, use of aZm miR167 tag in a transgene construct expressing anti-pathogenemolecules can prevent leaky expression of such molecules in the seeds.

3.1.1.5 Mycotoxin Reduction/Elimination

Production of mycotoxins, including aflatoxin and fumonisin, by fungiassociated with plants is a significant factor in rendering the grainnot useful. These fungal organisms do not cause disease symptoms and/orinterfere with the growth of the plant, but they produce chemicals(mycotoxins) that are toxic to animals. Inhibition of the growth ofthese fungi would reduce the synthesis of these toxic substances and,therefore, reduce grain losses due to mycotoxin contamination. Novelgenes may be introduced into plants that would inhibit synthesis of themycotoxin without interfering with fungal growth. Expression of a novelgene which encodes an enzyme capable of rendering the mycotoxin nontoxicwould be useful in order to achieve reduced mycotoxin contamination ofgrain. The result of any of the above mechanisms would be a reducedpresence of mycotoxins on grain.

For this application either a miRNA-tag, which allows for enhancedspecific expression in tissue, which presents the interaction or entryside for the fungal pathogen (e.g., the epidermis) or a miRNA-tagcorresponding to an miRNA, which is endogenously suppressed by thefungal pathogen induced stress factor is preferred to be employed fordesigning the miRNA-tag. Alternatively, a miRNA-tag, which ensuresenhanced seed-specific or preferential expression can be employed. Forexample, maize miR156 is expressed everywhere but seeds, use of miR156tag could enhance seed-specific expression.

3.1.1.6 Grain Composition or Quality

Genes may be introduced into plants, particularly commercially importantcereals such as maize, wheat or rice, to improve the grain for which thecereal is primarily grown. A wide range of novel transgenic plantsproduced in this manner may be envisioned depending on the particularend use of the grain.

For example, the largest use of maize grain is for feed or food.Introduction of genes that alter the composition of the grain maygreatly enhance the feed or food value. The primary components of maizegrain are starch, protein, and oil. Each of these primary components ofmaize grain may be improved by altering its level or composition.Several exampies may be mentioned for illustrative purposes but in noway provide an exhaustive list of possibilities.

The protein of many cereal grains is suboptimal for feed and foodpurposes especially when fed to pigs, poultry, and humans. The proteinis deficient in several amino acids that are essential in the diet ofthese species, requiring the addition of supplements to the grain.Limiting essential amino acids may include lysine, methionine,tryptophan, threonine, valine, arginine, and histidine. Some amino acidsbecome limiting only after the grain is supplemented with other inputsfor feed formulations. For example, when the grain is supplemented withsoybean meal to meet lysine requirements, methionine becomes limiting.The levels of these essential amino acids in seeds and grain may beelevated by mechanisms which include, but are not limited to, theintroduction of genes to increase the biosynthesis of the amino acids,decrease the degradation of the amino acids, increase the storage of theamino acids in proteins, or increase transport of the amino acids to theseeds or grain.

One mechanism for increasing the biosynthesis of the amino acids is tointroduce genes that deregulate the amino acid biosynthetic pathwayssuch that the plant can no longer adequately control the levels that areproduced. This may be done by deregulating or bypassing steps in theamino acid biosynthetic pathway which are normally regulated by levelsof the amino acid end product of the pathway. Examples include theintroduction of genes that encode deregulated versions of the enzymesaspartokinase or dihydrodipicolinic acid (DHDP)-synthase for increasinglysine and threonine production, and anthranilate synthase forincreasing tryptophan production. Reduction of the catabolism of theamino acids may be accomplished by introduction of DNA sequences thatreduce or eliminate the expression of genes encoding enzymes thatcatalyse steps in the catabolic pathways such as the enzymelysine-ketoglutarate reductase.

The protein composition of the grain may be altered to improve thebalance of amino acids in a variety of ways including elevatingexpression of native proteins, decreasing expression of those with poorcomposition, changing the composition of native proteins, or introducinggenes encoding entirely new proteins possessing superior composition.DNA may be introduced that decreases the expression of members of thezein family of storage proteins. This DNA may encode ribozymes orantisense sequences directed to impairing expression of zein proteins orexpression of regulators of zein expression such as the opaque-2 geneproduct. The protein composition of the grain may be modified throughthe phenomenon of cosuppression, i.e., inhibition of expression of anendogenous gene through the expression of an identical structural geneor gene fragment introduced through transformation (Goring 1991).Additionally, the introduced DNA may encode enzymes which degradezeines. The decreases in zein expression that are achieved may beaccompanied by increases in proteins with more desirable amino acidcomposition or increases in other major seed constituents such asstarch. Alternatively, a chimeric gene may be introduced that comprisesa coding sequence for a native protein of adequate amino acidcomposition such as for one of the globulin proteins or 10 kD zein ofmaize and a promoter or other regulatory sequence designed to elevateexpression of said protein. The coding sequence of said gene may includeadditional or replacement codons for essential amino acids. Further, acoding sequence obtained from another species, or, a partially orcompletely synthetic sequence encoding a completely unique peptidesequence designed to enhance the amino acid composition of the seed maybe employed.

The introduction of genes that alter the oil content of the grain may beof value. Increases in oil content may result in increases inmetabolizable energy content and density of the seeds for uses in feedand food. The introduced genes may encode enzymes that remove or reducerate-limitations or regulated steps in fatty acid or lipid biosynthesis.Such genes may include, but are not limited to, those that encodeacetyl-CoA carboxylase, ACPacyltransferase, 3-ketoacyl-ACP synthase,plus other well-known fatty acid biosynthetic activities. Otherpossibilities are genes that encode proteins that do not possessenzymatic activity such as acyl carrier protein. Additional examplesinclude 2-acetyltransferase, oleosin pyruvate dehydrogenase complex,acetyl CoA synthetase, ATP citrate lyase, ADP-glucose pyrophosphorylaseand genes of the carnitine-CoA-acetyl-CoA shuttles. It is anticipatedthat expression of genes related to oil biosynthesis will be targeted tothe plastid, using a plastid transit peptide sequence and preferablyexpressed in the seed embryo. Genes may be introduced that alter thebalance of fatty acids present in the oil providing a more healthful ornutritive feedstuff. The introduced DNA may also encode sequences thatblock expression of enzymes involved in fatty acid biosynthesis,altering the proportions of fatty acids present in the grain such asdescribed below.

Genes may be introduced that enhance the nutritive value of the starchcomponent of the grain, for example by increasing the degree ofbranching, resulting in improved utilization of the starch in cows bydelaying its metabolism.

Besides affecting the major constituents of the grain, genes may beintroduced that affect a variety of other nutritive, processing, orother quality aspects of the grain as used for feed or food. Forexample, pigmentation of the grain may be increased or decreased.Enhancement and stability of yellow pigmentation is desirable in someanimal feeds and may be achieved by introduction of genes that result inenhanced production of xanthophylls and carotenes by eliminatingrate-limiting steps in their production. Such genes may encode alteredforms of the enzymes phytoene synthase, phytoene desaturase, or lycopenesynthase. Alternatively, unpigmented white corn is desirable forproduction of many food products and may be produced by the introductionof DNA which blocks or eliminates steps in pigment production pathways.

Feed or food comprising some cereal grains possesses insufficientquantities of vitamins and must be supplemented to provide adequatenutritive value. Introduction of genes that enhance vitamin biosynthesisin seeds may be envisioned including, for example, vitamins A, E, B₁₂,choline, and the like. For example, maize grain also does not possesssufficient mineral content for optimal nutritive value. Genes thataffect the accumulation or availability of compounds containingphosphorus, sulfur, calcium, manganese, zinc, and iron among otherswould be valuable. An example may be the introduction of a gene thatreduced phytic acid production or encoded the enzyme phytase whichenhances phytic acid breakdown. These genes would increase levels ofavailable phosphate in the diet, reducing the need for supplementationwith mineral phosphate.

Numerous other examples of improvement of cereals for feed and foodpurposes might be described. The improvements may not even necessarilyinvolve the grain, but may, for example, improve the value of the grainfor silage. Introduction of DNA to accomplish this might includesequences that alter lignin production such as those that result in the“brown midrib” phenotype associated with superior feed value for cattle.

In addition to direct improvements in feed or food value, genes may alsobe introduced which improve the processing of grain and improve thevalue of the products resulting from the processing. The primary methodof processing certain grains such as maize is via wetmilling. Maize maybe improved though the expression of novel genes that increase theefficiency and reduce the cost of processing such as by decreasingsteeping time.

Improving the value of wetmilling products may include altering thequantity or quality of starch, oil, corn gluten meal, or the componentsof corn gluten feed. Elevation of starch may be achieved through theidentification and elimination of rate limiting steps in starchbiosynthesis or by decreasing levels of the other components of thegrain resulting in proportional increases in starch. An example of theformer may be the introduction of genes encoding ADP-glucosepyrophosphorylase enzymes with altered regulatory activity or which areexpressed at higher level. Examples of the latter may include selectiveinhibitors of, for example, protein or oil biosynthesis expressed duringlater stages of kernel development.

The properties of starch may be beneficially altered by changing theratio of amylose to amylopectin, the size of the starch molecules, ortheir branching pattern. Through these changes a broad range ofproperties may be modified which include, but are not limited to,changes in gelatinization temperature, heat of gelatinization, clarityof films and pastes, Theological properties, and the like. To accomplishthese changes in properties, genes that encode granule-bound or solublestarch synthase activity or branching enzyme activity may be introducedalone or combination. DNA such as antisense constructs may also be usedto decrease levels of endogenous activity of these enzymes. Theintroduced genes or constructs may possess regulatory sequences thattime their expression to specific intervals in starch biosynthesis andstarch granule development. Furthermore, it may be advisable tointroduce and express genes that result in the in vivo derivatization,or other modification, of the glucose moieties of the starch molecule.The covalent attachment of any molecule may be envisioned, limited onlyby the existence of enzymes that catalyze the derivatizations and theaccessibility of appropriate substrates in the starch granule. Examplesof important derivations may include the addition of functional groupssuch as amines, carboxyls, or phosphate groups which provide sites forsubsequent in vitro derivatizations or affect starch properties throughthe introduction of ionic charges. Examples of other modifications mayinclude direct changes of the glucose units such as loss of hydroxylgroups or their oxidation to aldehyde or carboxyl groups.

Oil is another product of wetmilling of corn and other grains, the valueof which may be improved by introduction and expression of genes. Thequantity of oil that can be extracted by wetmilling may be elevated byapproaches as described for feed and food above. Oil properties may alsobe altered to improve its performance in the production and use ofcooking oil, shortenings, lubricants or other oil-derived products orimprovement of its health attributes when used in the food-relatedapplications. Novel fatty acids may also be synthesized which uponextraction can serve as starting materials for chemical syntheses. Thechanges in oil properties may be achieved by altering the type, level,or lipid arrangement of the fatty acids present in the oil. This in turnmay be accomplished by the addition of genes that encode enzymes thatcatalyze the synthesis of novel fatty acids and the lipids possessingthem or by increasing levels of native fatty acids while possiblyreducing levels of precursors. Alternatively DNA sequences may beintroduced which slow or block steps in fatty acid biosynthesisresulting in the increase in precursor fatty acid intermediates. Genesthat might be added include desaturases, epoxidases, hydratases,dehydratases, and other enzymes that catalyze reactions involving fattyacid intermediates. Representative examples of catalytic steps thatmight be blocked include the desaturations from stearic to oleic acidand oleic to linolenic acid resulting in the respective accumulations ofstearic and oleic acids.

Improvements in the other major cereal wetmilling products, gluten mealand gluten feed, may also be achieved by the introduction of genes toobtain novel plants. Representative possibilities include but are notlimited to those described above for improvement of food and feed value.

In addition it may further be considered that the plant be used for theproduction or manufacturing of useful biological compounds that wereeither not produced at all, or not produced at the same level, in theplant previously. The novel plants producing these compounds are madepossible by the introduction and expression of genes by transformationmethods. The possibilities include, but are not limited to, anybiological compound which is presently produced by any organism such asproteins, nucleic acids, primary and intermediary metabolites,carbohydrate polymers, etc. The compounds may be produced by the plant,extracted upon harvest and/or processing, and used for any presentlyrecognized useful purpose such as pharmaceuticals, fragrances,industrial enzymes to name a few.

Further possibilities to exemplify the range of grain traits orproperties potentially encoded by introduced genes in transgenic plantsinclude grain with less breakage susceptibility for export purposes orlarger grit size when processed by dry milling through introduction ofgenes that enhance gamma-zein synthesis, popcorn with improved popping,quality and expansion volume through genes that increase pericarpthickness, corn with whiter grain for food uses though introduction ofgenes that effectively block expression of enzymes involved in pigmentproduction pathways, and improved quality of alcoholic beverages orsweet corn through introduction of genes which affect flavor such as theshrunken gene (encoding sucrose synthase) for sweet corn.

For seed-based applications, a miRNA-tag, which ensures enhancedseed-specific or preferential expression can be employed.

3.1.1.7 Tuber or Seed Composition or Quality

Various traits can be advantegously expressed especially in seeds ortubers to improve composition or quality. Such traits include but arenot lifted to:

-   -   Expression of metabolic enzymes for use in the food-and-feed        sector, for example of phytases and cellulases. Especially        preferred are nucleic acids such as the artificial cDNA which        encodes a microbial phytase (GenBank Acc. No.: A19451) or        functional equivalents thereof.    -   Expression of genes which bring about an accumulation of fine        chemicals such as of tocopherols, tocotrienols or carotenoids.        An example is phytoene desaturase. Preferred are nucleic acids        which encode the Narcissus pseudonarcissus photoene desaturase        (GenBank Acc. No.: X78815) or functional equivalents thereof.    -   Production of nutraceuticals such as, for example,        polyunsaturated fatty acids (for example arachidonic acid,        eicosapentaenoic acid or docosahexaenoic acid) by expression of        fatty acid elongases and/or desaturases, or production of        proteins with improved nutritional value such as, for example,        with a high content of essential amino acids (for example the        high-methionine 2S albumin gene of the brazil nut). Preferred        are nucleic acids which encode the Bertholletia excelsa        high-methionine 2S albumin (GenBank Acc. No.: AB044391), the        Physcomitrella patens Δ6-acyl-lipid desaturase (GenBank Acc.        No.: AJ222980; Girke et al. (1998) Plant J 15:39-48), the        Mortierella alpina Δ6-desaturase (Sakuradani et al. 1999 Gene        238:445-453), the Caenorhabditis elegans Δ5-desaturase        (Michaelson et al. 1998, FEBS Letters 439:215-218), the        Caenorhabditis elegans Δ5-fatty acid desaturase (des-5) (GenBank        Acc. No.: AF078796), the Mortierella alpina Δ5-desaturase        (Michaelson et al. JBC 273:19055-19059), the Caenorhabditis        elegans Δ6-elongase (Beaudoin et al. 2000, PNAS 97:6421-6426),        the Physcomitrella patens Δ6-elongase (Zank et al. 2000,        Biochemical Society Transactions 28:654-657), or functional        equivalents of these.    -   Production of high-quality proteins and enzymes for industrial        purposes (for example enzymes, such as lipases) or as        pharmaceuticals (such as, for example, antibodies, blood        clotting factors, interferons, lymphokins, colony stimulation        factor, plasminogen activators, hormones or vaccines, as        described by Hood E E, Jilka J M (1999) Curr Opin Biotechnol        10(4):382-6; Ma J K, Vine N D (1999) Curr Top Microbiol Immunol        236:275-92). For example, it has been possible to produce        recombinant avidin from chicken albumen and bacterial        b-glucuronidase (GUS) on a large scale in transgenic maize        plants (Hood et al. (1999) Adv Exp Med Biol 464:127-47. Review).    -   Obtaining an increased storability in cells which normally        comprise fewer storage proteins or storage lipids, with the        purpose of increasing the yield of these substances, for example        by expression of acetyl-CoA carboxylase. Preferred nucleic acids        are those which encode the Medicago sativa acetyl-CoA        carboxylase (ACCase) (GenBank Acc. No.: L25042), or functional        equivalents thereof.    -   Reducing levels of α-glucan L-type tuber phosphorylase (GLTP) or        .α-glucan H-type tuber phosphorylase (GHTP) enzyme activity        preferably within the potato tuber (see U.S. Pat. No.        5,998,701). The conversion of starches to sugars in potato        tubers, particularly when stored at temperatures below 7° C., is        reduced in tubers exhibiting reduced GLTP or GHTP enzyme        activity. Reducing cold-sweetening in potatoes allows for potato        storage at cooler temperatures, resulting in prolonged dormancy,        reduced incidence of disease, and increased storage life.        Reduction of GLTP or GHTP activity within the potato tuber may        be accomplished by such techniques as suppression of gene        expression using homologous antisense or double-stranded RNA,        the use of co-suppression, regulatory silencing sequences. A        potato plant having improved cold-storage characteristics,        comprising a potato plant transformed with an expression        cassette having a TPT promoter sequence operably linked to a DNA        sequence comprising at least 20 nucleotides of a gene encoding        an α-glucan phosphorylase selected from the group consisting of        α-glucan L-type tuber phosphorylase (GLTP) and α-glucan H-type        phosphorylase (GHTP).

Further examples of advantageous genes are mentioned for example inDunwell J M, Transgenic approaches to crop improvement, J Exp Bot. 2000;51 Spec No; pages 487-96.

For seed-based applications, a miRNA-tag, which ensures enhanced seed ortuber-specific or preferential expression can be employed. For example,maize miR156 is expressed everywhere but seeds, use of miR156 tag couldenhance seed-specific expression.

3.1.1.8 Plant Agronomic Characteristics

Two of the factors determining where plants can be grown are the averagedaily temperature during the growing season and the length of timebetween frosts. Within the areas where it is possible to grow aparticular plant, there are varying limitations on the maximal time itis allowed to grow to maturity and be harvested. The plant to be grownin a particular area is selected for its ability to mature and dry downto harvestable moisture content within the required period of time withmaximum possible yield. Therefore, plant of varying maturities aredeveloped for different growing locations. Apart from the need to drydown sufficiently to permit harvest is the desirability of havingmaximal drying take place in the field to minimize the amount of energyrequired for additional drying post-harvest. Also the more readily thegrain can dry down, the more time there is available for growth andkernel fill. Genes that influence maturity and/or dry down can beidentified and introduced into plant lines using transformationtechniques to create new varieties adapted to different growinglocations or the same growing location but having improved yield tomoisture ratio at harvest. Expression of genes that are involved inregulation of plant development may be especially useful, e.g., theliguleless and rough sheath genes that have been identified in plants.

Genes may be introduced into plants that would improve standability andother plant growth characteristics. For example, expression of novelgenes which confer stronger stalks, improved root systems, or prevent orreduce ear droppage would be of great value to the corn farmer.Introduction and expression of genes that increase the total amount ofphotoassimilate available by, for example, increasing light distributionand/or interception would be advantageous. In addition the expression ofgenes that increase the efficiency of photosynthesis and/or the leafcanopy would further increase gains in productivity. Such approacheswould allow for increased plant populations in the field.

Delay of late season vegetative senescence would increase the flow ofassimilate into the grain and thus increase yield. Overexpression ofgenes within plants that are associated with “stay green” or theexpression of any gene that delays senescence would be advantageous. Forexample, a non-yellowing mutant has been identified in Festuca pratensis(Davies 1990). Expression of this gene as well as others may preventpremature breakdown of chlorophyll and thus maintain canopy function.

3.1.1.9 Nutrient Utilization

The ability to utilize available nutrients and minerals may be alimiting factor in growth of many plants. It is proposed that it wouldbe possible to alter nutrient uptake, tolerate pH extremes, mobilizationthrough the plant, storage pools, and availability for metabolicactivities by the introduction of novel genes. These modifications wouldallow a plant to more efficiently utilize available nutrients. It iscontemplated that an increase in the activity of, for example, an enzymethat is normally present in the plant and involved in nutrientutilization would increase the availability of a nutrient. An example ofsuch an enzyme would be phytase. It is also contemplated that expressionof a novel gene may make a nutrient source available that was previouslynot accessible, e.g., an enzyme that releases a component of nutrientvalue from a more complex molecule, perhaps a macromolecule.

For seed-based applications, a miRNA-tag, which ensures enhancedseed-specific or preferential expression can be employed. For example,maize miR156 is expressed everywhere but seeds, use of miR156 tag couldenhance seed-specific expression.

3.1.1.10 Male Sterility

Male sterility is useful in the production of hybrid seed. It isproposed that male sterility may be produced through expression of novelgenes. For example, it has been shown that expression of genes thatencode proteins that interfere with development of the maleinflorescence and/or gametophyte result in male sterility. Chimericribonuclease genes that express in the anthers of transgenic tobacco andoilseed rape have been demonstrated to lead to male sterility (Mariani1990). For example, a number of mutations were discovered in maize thatconfers cytoplasmic male sterility. One mutation in particular, referredto as T cytoplasm, also correlates with sensitivity to Southern cornleaf blight. A DNA sequence, designated TURF-13 (Levings 1990), wasidentified that correlates with T cytoplasm. It would be possiblethrough the introduction of TURF-13 via transformation to separate malesterility from disease sensitivity. As it is necessary to be able torestore male fertility for breeding purposes and for grain production,it is proposed that genes encoding restoration of male fertility mayalso be introduced.

For this application, a miRNA-tag, which ensures enhancedpollen-specific or preferential expression can be employed.

3.1.2 Plant Target Genes for Gene Silencing with Enhanced Specificity

DNA may be introduced into plants for the purpose of expressing RNAtranscripts that function to affect plant phenotype yet are nottranslated into protein. Two examples are antisense RNA and RNA withribozyme activity. Both may serve possible functions in reducing oreliminating expression of native or introduced plant genes.

Genes may be constructed or isolated, which when transcribed, produceantisense RNA or double-stranded RNA that is complementary to all orpart(s) of a targeted messenger RNA(s). The antisense RNA reducesproduction of the polypeptide product of the messenger RNA. Thepolypeptide product may be any protein encoded by the plant genome. Theaforementioned genes will be referred to as antisense genes. Anantisense gene may thus be introduced into a plant by transformationmethods to produce a novel transgenic plant with reduced expression of aselected protein of interest. For example, the protein may be an enzymethat catalyzes a reaction in the plant. Reduction of the enzyme activitymay reduce or eliminate products of the reaction which include anyenzymatically synthesized compound in the plant such as fatty acids,amino acids, carbohydrates, nucleic acids and the like. Alternatively,the protein may be a storage protein, such as a zein, or a structuralprotein, the decreased expression of which may lead to changes in seedamino acid composition or plant morphological changes respectively. Thepossibilities cited above are provided only by way of example and do notrepresent the full range of applications.

Expression of antisense-RNA or double-stranded RNA by one of theexpression cassettes of the invention is especially preferred. Alsoexpression of sense RNA can be employed for gene silencing(co-suppression). This RNA is preferably a non-translatable RNA. Generegulation by double-stranded RNA (“double-stranded RNA interference”;dsRNAi) is well known in the arte and described for various organismincluding plants (e.g., Matzke 2000; Fire A et al 1998; WO 99/32619; WO99/53050; WO 00/68374; WO 00/44914; WO 00/44895; WO 00/49035; WO00/63364).

Genes may also be constructed or isolated, which when transcribedproduce RNA enzymes, or ribozymes, which can act as endoribonucleasesand catalyze the cleavage of RNA molecules with selected sequences. Thecleavage of selected messenger RNA's can result in the reducedproduction of their encoded polypeptide products. These genes may beused to prepare novel transgenic plants which possess them. Thetransgenic plants may possess reduced levels of polypeptides includingbut not limited to the polypeptides cited above that may be affected byantisense RNA.

It is also possible that genes may be introduced to produce noveltransgenic plants which have reduced expression of a native gene productby a mechanism of cosuppression. It has been demonstrated in tobacco,tomato, and petunia (Goring 1991; Smith 1990; Napoli 1990; van der Krol1990) that expression of the sense transcript of a native gene willreduce or eliminate expression of the native gene in a manner similar tothat observed for antisense genes. The introduced gene may encode all orpart of the targeted native protein but its translation may not berequired for reduction of levels of that native protein.

The possible target genes stated are to be understood by way of example,but not by limitation:

3.1.2.1 Improved protection against abiotic stress factors (heat, chill,drought, increased moisture, environmental toxins, UV radiation). It ispreferred to reduce the expression of genes, which are involved instress reactions.

-   -   For this application either a miRNA-tag, which allows for        enhanced specific expression in sensitive tissue (young        seedling, embryo) or a miRNA-tag corresponding to an miRNA,        which is endogenously suppressed by the stress factor is        preferred to be employed for designing the miRNA-tag.

3.1.2.2 Modification of the Composition and/or the Content of FattyAcids, Lipids or Oils

A modification of the fatty acid contents or the fatty acid composition,preferably in an oil crop such as oilseed rape or sunflower, can beachieved, for example, by reducing the gene expression of fatty acidbiosynthesis genes, preferably those selected from the group consistingof genes encoding acetyl transacylases, acyl transport proteins (“acylcarrier protein”), desaturases such as stearyl desaturases or microsomalD12-desaturases, in particular Fad2-1 genes, malonyl transacylase,β-ketoacyl-ACP synthetases, 3-keto-ACP reductases, enoyl-ACP hydrases,thioesterases such as acyl-ACP thioesterases, enoyl-ACP reductases.Various further advantageous approaches for modifying the lipidcomposition are described (Shure M et al. (1983) Cell 35:225-233; Preisset al. (1987) Tailoring Genes for Crop Improvement (Bruening et al.,eds.), Plenum Press, S. 133-152; Gupta et al. (1988) Plant Mol Biol.10:215-224; Olive et al. (1989) Plant Mol Biol 12:525-538; Bhattacharyyaet al. (1990) Cell 60:155-122; Dunwell J M (2000) J Exp Botany 51SpecNo:487-96; Brar D S et al. (1996) Biotech Genet Eng Rev 13:167-79;Kishore G M and Somerville C R (1993) Curr Opin Biotech 4(2):152-8).Preferred are, in particular, Fad2 genes (for example those described byGenbank Acc. No.: AF124360 (Brassica carinata), AF042841 (Brassicarapa), L26296 (Arabidopsis thaliana), A65102 (Corylus avellana)).Further advantageous genes and methods for modifying the lipid contentare described, for example, in U.S. Pat. No. 5,530,192 and WO 94/18337.Elevated lipid content can also be achieved by reducing the starchcontent, for example as the result of the reduced expression of enzymesof the carbohydrate metabolism (for example ADP-glucosepyrophosphorylases).

For this application either a miRNA-tag, which allows for enhancedspecific expression in seeds is preferred for designing the miRNA-tag.For example, maize miR156 is expressed everywhere but seeds, use ofmiR156 tag could enhance seed-specific expression.

3.1.2.3 Modification of the Carbohydrate Composition

A modification of the carbohydrate composition can be achieved forexample by reducing the gene expression of carbohydrate metabolism genesor of carbohydrate biosynthesis genes, for example genes of thebiosynthesis of amylose, pectins, cellulose or cell wall carbohydrates.A multiplicity of cellular processes (maturation, storability, starchcomposition or starch content and the like) can thereby be influenced inan advantageous manner. Target genes which may be mentioned by way ofexample, but not by limitation, are phosphorylases, starch synthetases,0-enzymes, sucrose-6-phosphate synthetases, sucrose-6-phosphatephosphatases, ADP-glucose pyrophosphorylases, branching enzymes,debranching enzymes and various amylases. The corresponding genes aredescribed (Dunwell J M (2000) J Exp Botany 51Spec No:487-96; Brar D S etal. (1996) Biotech Genet Eng Rev 13:167-79; Kishore G M and Somerville CR (1993) Curr Opin Biotech 4(2):152-8). Advantageous genes forinfluencing the carbohydrate metabolism—in particular starchbiosynthesis—are described in WO 92/11375, WO 92/11376, U.S. Pat. No.5,365,016 and WO 95/07355.

For this application either a miRNA-tag, which allows for enhancedspecific expression in seeds is preferred for designing the miRNA-tag.For example, maize miR156 is expressed everywhere but seeds, use ofmiR156 tag could enhance seed-specific expression.

3.1.2.4 Modification of the Color or Pigmentation

A modification of the color or pigmentation, preferably of ornamentals,can be achieved for example by reducing the gene expression of flavonoidbiosynthesis genes such as, for example, the genes of chalconesynthases, chalcone isomerases, phenylalanine ammonia lyases,dehydrokaempferol (flavone) hydroxylases such as flavanone3-hydroxylases or flavanone 2-hydroxylases, dihydroflavonol reductases,dihydroflavanol 2-hydroxylases, flavonoid 3′-hydroxylases, flavonoid5′-hydroxylases, flavonoid glycosyltransferases (for exampleglucosyltransferases such as UDPG:flavonoid 3-O-glucosyltransferases,UDPG:flavonol 7-O-glucosyltransferases or rhamnosyltransferases),flavonoid methyltransferases (such as, for example,SAM:anthocyanidin-3-(p-coumaroyl)rutinoside-5-glucoside-3′,5′-O-methyltransferases)and flavonoid acyltransferases (Hahlbrock (1981) Biochemistry of Plants,Vol. 7, Conn (Ed.); Weiring and de Vlaming (1984) “Petunia”, K C Sink(Ed.), Springer-Verlag, New York). Particularly suitable are thesequences described in EP-A1 522 880.

For this application either a miRNA-tag, which allows for enhancedspecific expression in flowers and its part is preferred for designingthe miRNA-tag. For example, rice miR156I is expressed in root and shoot,use of miR156I tag can enhance specific expression of gene-of-interestin flowers.

3.1.2.5. Reduction of the Storage Protein Content

The reduction of the gene expression of genes encoding storage proteins(SP hereinbelow) has a large number of advantages such as, for example,the reduction of the allergenic potential or modification in thecomposition or quantity of other metabolites. Storage proteins aredescribed, inter alia, in EP-A 0 591 530, WO 87/47731, WO 98/26064, EP-A0 620 281; Kohno-Murase J et al. (1994) Plant Mol Biol 26(4): 1115-1124.SP serve for the storage of carbon, nitrogen and sulfur, which arerequired for the rapid heterotrophic growth in the germination of seedsor pollen. In most cases, they have no enzymatic activity. SP aresynthesized in the embryo only during seed development and, in thisprocess, accumulate firstly in protein storage vacuoles (PSV) ofdifferently differentiated cells in the embryo or endosperm. Storageproteins can be classified into subgroups, as the function of furthercharacteristic properties, such as, for example, their sedimentationcoefficient or the solubility in different solutions (water, saline,alcohol). The sedimentation coefficient can be determined by means ofultracentrifugation in the manner with which the skilled worker isfamiliar (for example as described in Correia J J (2000) Methods inEnzymology 321:81-100). In total, four large gene families for storageproteins can be assigned, owing to their sequences: 2S albumins(napin-like), 7S globulins (phaseolin-like), 11S/12S globulins(legumin/cruciferin-like) and the zein prolamins.

2S albumins are found widely in seeds of dicots, including importantcommercial plant families such as Fabaceae (for example soybean),Brassicaceae (for example oilseed rape), Euphorbiaceae (for examplecastor-oil plant) or Asteraceae (for example sunflower). 2S albumins arecompact globular proteins with conserved cysteine residues whichfrequently form heterodimers. 7S globulins occur in trimeric form andcomprise no cysteine residues. After their synthesis, they are cleavedinto smaller fragments and glycosylated, as is the case with the 2Salbumins. Despite differences in polypeptide size, the different 7Sglobulins are highly conserved and can probably be traced to a sharedprecursor protein, as is the case with the 2S albumins. Only smallamounts of the 7S globulins are found in monocots. In dicots, theyalways amount to less than the 11S/12S globulins. 11S/12S globulinsconstitute the main fraction of the storage proteins in dicots, inaddition to the 2S albumins. The high degree of similarity of thedifferent 11S globulins from different plant genera, in turn, allow theconclusion of a shared precursor protein in the course of evolution. Thestorage protein is preferably selected from the classes of the 2Salbumins (napin-like), 7S globulins (phaseolin-like), 11S/12S globulins(legumin/cruciferin-like) or zein prolamins. Especially preferred11S/12S globulins comprise preferably 11S globulins from oilseed rape,soybean and Arabidopsis, sunflower, linseed, sesame, safflower, olivetree, soybean or various nut species. Especially preferred zeinprolamins preferably comprise those from monocotyledonous plants, inparticular maize, rice, oats, barley or wheat.

For this application either a miRNA-tag, which allows for enhancedspecific expression in seeds is preferred for designing the miRNA-tag.For example, maize miR156 is expressed everywhere but seeds, use ofmiR156 tag could enhance seed-specific expression.

3.1.2.6. Obtaining a Resistance to Plant Pathogens

The methods and means of the invention will be especially suited forobtaining pathogen (e.g., virus or nematode) resistance, in eukaryoticcells or organisms, particularly in plant cells and plants. It isexpected that the chimeric RNA molecules (or the dsRNA molecules derivedtherefrom) produced by transcription in a host organism (e.g., a plant),can spread systemically throughout the organism. Thus it is possible toreduce the phenotypic expression of a nucleic acid in cells of anon-transgenic scion of a plant grafted onto a transgenic stockcomprising the chimeric genes of the invention (or vice versa) a methodwhich may be important in horticulture, viticulture or in fruitproduction.

A resistance to plant pathogens such as arachnids, fungi, insects,nematodes, protozoans, viruses, bacteria and diseases can be achieved byreducing the gene expression of genes which are essential for thegrowth, survival, certain developmental stages (for example pupation) orthe multiplication of a certain pathogen. A suitable reduction can bringabout a complete inhibition of the above steps, but also a delay ofsame. This may be plant genes which, for example, allow the pathogen toenter, but may also be pathogenhomologous genes. Preferably, thechimeric RNA (or the dsRNA derived therefrom) is directed against genesof the pathogen. For example, plants can be treated with suitableformulations of abovementioned agents, for example sprayed or dusted,the plants themselves, however, may also comprise the agents in the formof a transgenic organism and pass them on to the pathogens, for examplein the form of a stomach poison. Various essential genes of a variety ofpathogens are known to the skilled worker (for example for nematoderesistance: WO 93/10251, WO 94/17194).

Thus, an aspect of this invention provides a method where the targetgene for suppression encodes a protein in a plant pathogen (e.g., aninsect or nematode). In an aspect, a method comprises introducing intothe genome of a pathogen-targeted plant a nucleic acid constructcomprising DNA which is transcribed into a chimeric RNA that forms atleast one dsRNA molecule which is effective for reducing expression of atarget gene within the pathogen when the pathogen (e.g., insect ornematode) ingests or infects cells from said plant. In a preferredembodiment, the gene suppression is fatal to the pathogen.

Most preferred as pathogen are fungal pathogens such as Phytophthorainfestans, Fusarium nivale, Fusarium graminearum, Fusarium culmorum,Fusarium oxysporum, Blumeria graminis, Magnaporthe grisea, Sclerotiniasclerotium, Septoria nodorum, Septoria tritici, Alternaria brassicae,Phoma lingam, bacterial pathogens such as Corynebacterium sepedonicum,Erwinia carotovora, Erwinia amylovora, Streptomyces scabies, Pseudomonassyringae pv. tabaci, Pseudomonas syringae pv. phaseolicola, Pseudomonassyringae pv. tomato, Xanthomonas campestris pv. malvacearum andXanthomonas campestris pv. oryzae, and nematodes such as Globoderarostochiensis, G. paffida, Heterodera schachtii, Heterodera avenae,Ditylenchus dipsaci, Anguina tritici and Meloidogyne hapla.

Resistance to viruses can be obtained for example by reducing theexpression of a viral coat protein, a viral replicase, a viral proteaseand the like. A large number of plant viruses and suitable target genesare known to the skilled worker. The methods and compositions of thepresent invention are especially useful to obtain nematode resistantplants (for target genes see e.g., WO 92/21757, WO 93/10251, WO94/17194).

Also provided by the invention is a method for obtaining pathogenresistant organisms, particularly plants, comprising the steps ofproviding cells of the organism with an chimeric RNA molecule of theinvention, said chimeric RNA molecule capable to provide in aneukaryotic cell an at least partially double-stranded RNA molecule, saidchimeric RNA molecule comprising

-   a) at least one first ribonucleotide sequence that is substantially    identical to at least a part of a target nucleotide sequence of at    least one gene of a pathogen, and-   b) at least one second ribonucleotide sequence which is    substantially complementary to said first nucleotide sequence and is    capable to hybridizes to said first nucleotide sequence to form a    double-stranded RNA structure, and-   c) at least one third ribonucleotide sequence located between said    first and said second ribonucleotide sequence comprising at least    one removable RNA element, which can be removed by the RNA    processing mechanism of an eukaryotic cell without subsequently    covalently joining the resulting sequences comprising said first and    said secand ribonucleotide sequence, respectively.

Preferably, said first ribonucleotide sequence has between 65 and 100%sequence identity, preferably, between 75 and 100%, more preferablybetween 85 and 100%, most preferably between 95 and 100%, with at leastpart of the nucleotide sequence of the genome of a pathogen. Morepreferably the pathogen is selected from the group of virus, bacteria,fungi, and nematodes.

For this application either a miRNA-tag, which allows for enhancedspecific expression in tissue, which functions as entry-site for thepathogen (e.g., epidermis) or a miRNA-tag corresponding to an miRNA,which is endogenously suppressed by the pathogen-induced stress ispreferred to be employed for designing the miRNA-tag.

3.1.2.7. Prevention of Stem Break

A reduced susceptibility to stem break can be obtained for example byreducing the gene expression of genes of the carbohydrate metabolism(see above). Advantageous genes are described (WO 97/13865, inter alia)and comprise tissue-specific polygalacturonases or cellulases.

For this application either a miRNA-tag, which allows for enhancedspecific expression in stem is preferred for designing the miRNA-tag.For example, maize miR166 is expressed in leafs and tassel, use ofmiR166 tag can enhance specific expression of gene-of-interest in stem.

3.1.2.8. Delay of Fruit Maturation

Delayed fruit maturation can be achieved for example by reducing thegene expression of genes selected from the group consisting ofpolygalacturonases, pectin esterases, β-(1-4)glucanases (cellulases),β-galactanases (β-galactosidases), or genes of ethylene biosynthesis,such as 1-aminocyclopropane-1-carboxylate synthase, genes of carotenoidbiosynthesis such as, for example, genes of prephytoene or phytoenebiosynthesis, for example phytoene desaturases. Further advantageousgenes are, for example, in WO 91/16440, WO 91/05865, WO 91/16426, WO92/17596, WO 93/07275 or WO 92/04456, U.S. Pat. No. 5,545,366).

For this application either a miRNA-tag, which allows for enhancedspecific expression in fruits is preferred for designing the miRNA-tag.

3.1.2.9. Achieving male sterility. Suitable target genes are describedin WO 94/29465, WO89/10396, WO 92/18625, inter alia. A particularapplication for reduction of the phenotypic expression of a transgene ina plant cell, inter alia, has been described for the restoration of malefertility, the latter being obtained by introduction of a transgenecomprising a male sterility DNA (WO 94/09143, WO 91/02069). The nucleicacid of interest is specifically the male sterility DNA.

-   -   For this application either a miRNA-tag, which allows for        enhanced specific expression in pollen is preferred for        designing the miRNA-tag.

3.1.2.10. Reduction of undesired or toxic plant constituents such as,for example, glucosinolates. Suitable target genes are described (in WO97/16559, inter alia). For this application either a miRNA-tag, whichallows for enhanced specific expression in seeds is preferred fordesigning the miRNA-tag. For example, maize miR156 is expressedeverywhere but seeds, use of miR156 tag could enhance seed-specificexpression.

3.1.2.11. Delay of senescence symptoms. Suitable target genes are, interalia, cinnamoyl-CoA:NADPH reductases or cinnamoyl alcoholdehydrogenases. Further target genes are described (in WO 95/07993,inter alia).

3.1.2.12. Modification of the lignification and/or the lignin content,mainly in tree species. Suitable target genes are described in WO93/05159, WO 93/05160, inter alia.

3.1.2.13. Modification of the fiber content in foodstuffs, preferably inseeds, by reducing the expression of coffeic acid O-methyltransferase orof cinnamoyl alcohol dehydrogenase.

3.1.2.14. Modification of the fiber quality in cotton. Suitable targetgenes are described in U.S. Pat. No. 5,597,718, inter alia.

3.1.2.15. Reduction of the susceptibility to bruising of, for example,potatoes by reducing for example polyphenol oxidase (WO 94/03607) andthe like.

3.1.2.16. Enhancement of vitamin E biosynthesis, for example by reducingthe expression of genes from the homogentisate catabolic pathway suchas, for example, homogentisate 1,2-dioxygenase (HGD; EC No.: 1.13.11.5),maleylacetoacetate isomerase (MAAI; EC No.: 5.2.1.2.) orfumaryl-acetoacetate hydrolase (FAAH; EC No.: 3.7.1.2).

3.1.2.17. Reduction of the nicotine content for example in tobacco byreduced expression of, for example, N-methyl-putrescin oxidase andputrescin N-methyltransferase.

3.1.2.18. Reduction of the caffeine content in coffee bean (e.g., Coffeaarabica) by reducing the gene expression of genes of caffeinebiosynthesis such as 7-methylxanthine 3-methyltransferase.

3.1.2.19. Reduction of the theophylline content in tea (Camelliasinensis) by reducing the gene expression of genes of theophyllinebiosynthesis such as, for example, 1-methylxanthine 3-methyltransferase.

3.1.2.20. Increase of the methionine content by reducing threoninebiosynthesis, for example by reducing the expression of threoninesynthase (Zeh M et al. (2001) Plant Physiol 127(3):792-802).

Furthermore the method and compounds of the invention can be used forobtaining shatter resistance (WO 97/13865), for obtaining modifiedflower color patterns (EP 522 880, U.S. Pat. No. 5,231,020), forreducing the presence of unwanted (secondary) metabolites in organisms,such as glucosinolates (WO97/16559) or chlorophyll content (EP 779 364)in plants, for modifying the profile of metabolites synthesized in aeukaryotic cell or organisms by metabolic engineering e.g. by reducingthe expression of particular genes involved in carbohydrate metabolism(WO 92/11375, WO 92/11376, U.S. Pat. No. 5,365,016, WO 95/07355) orlipid biosynthesis (WO 94/18337, U.S. Pat. No. 5,530,192) etc. Furtherexamples of advantageous genes are mentioned for example in Dunwell J M,Transgenic approaches to crop improvement, J Exp Bot. 2000; 51 Spec No;pages 487-96.

Each of the abovementioned applications can be used as such on its own.Naturally, it is also possible to use more than one of theabovementioned approaches simultaneously. If, in this context, allapproaches are used, the expression of at least two differing targetgenes as defined above is reduced. In this context, these target genescan originate from a single group of genes which is preferred for a use,or else be assigned to different use groups.

3.1.3 Plant Transformation & Expression Technology

A chimeric RNA of the invention can be expressed within a plant cellusing conventional recombinant DNA technology. Generally, this involvesinserting a nucleotide sequence encoding the chimeric RNA of theinvention into an expression construct or expression vector usingstandard cloning procedures known in the art.

3.1.3.1. Requirements for Construction of Plant Expression Constructs

The expression construct or expression construct of the inventioncomprises one or more genetic control sequences (or regulatorysequences) operably linked to a nucleic acid sequence encoding thechimeric RNA of the invention. These genetic control sequences regulateexpression of the chimeric RNA in host cells. Genetic control sequencesare described, for example, in “Goeddel; Gene Expression Technology:Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)” or“Gruber and Crosby, in: Methods in Plant Molecular Biology andBiotechnolgy, CRC Press, Boca Raton, Fla., eds.: Glick and Thompson,Chapter 7, 89-108” and the references cited therein. Sequences intendedfor expression in plants are first operatively linked to a suitablepromoter functional in plants. Such expression constructs optionallycomprise further sequences required or selected for the expression ofthe transgene. Such sequences include, but are not restricted to,transcription terminators, extraneous sequences to enhance expression.These expression constructs are easily transferred to the planttransformation vectors described infra.

3.1.3.1.1. Promoters

The nucleic acid sequence encoding the chimeric RNA of the invention ispreferably operably linked to a plant-specific promoter. The term“plant-specific promoter” means principally any promoter which iscapable of governing the expression of genes, in particular foreignnucleic acid sequences or genes, in plants or plant parts, plant cells,plant tissues, plant cultures. In this context, the expressionspecificity of said plant-specific promoter can be for exampleconstitutive, tissue-specific, inducible or development-specific. Thefollowing are preferred:

3.1.3.1.1.1 Constitutive Promoters

Where expression of a gene in all tissues of a transgenic plant or otherorganism is desired, one can use a “constitutive” promoter, which isgenerally active under most environmental conditions and states ofdevelopment or cell differentiation. Useful promoters for plants alsoinclude those obtained from Ti- or Ri-plasmids, from plant cells, plantviruses or other organisms whose promoters are found to be functional inplants. Bacterial promoters that function in plants, and thus aresuitable for use in the methods of the invention include the octopinesynthetase promoter, the nopaline synthase promoter, and the mannopinesynthetase promoter. The promoter controlling expression of the chimericRNA of the invention (and/or selection marker) can be constitutive.Suitable constitutive promoters for use in plants include, for example,the cauliflower mosaic virus (CaMV) 35S transcription initiation region(Franck et al. (1980) Cell 21:285-294; Odell et al. (1985) Nature313:810-812; Shewmaker et al. (1985) Virology 140:281-288; Gardner etal. (1986) Plant Mol Biol 6:221-228), the 19S transcription initiationregion (U.S. Pat. No. 5,352,605 and WO 84/02913), and region VIpromoters, the 1′- or 2′-promoter derived from T-DNA of Agrobacteriumtumefaciens, and other promoters active in plant cells that are known tothose of skill in the art. Other suitable promoters include thefull-length transcript promoter from Figwort mosaic virus, actinpromoters (e.g., the rice actin promoter; McElroy et al. (1990) PlantCell 2: 163-171), histone promoters, tubulin promoters, or the mannopinesynthase promoter (MAS). Other constitutive plant promoters includevarious ubiquitin or poly-ubiquitin promoters (Sun and Callis (1997)Plant J 11(5): 1017-1027, Cristensen et al. (1992) Plant Mol Biol18:675-689; Christensen et al. (1989) Plant Mol. Biol. 12: 619-632;Bruce et al. (1989) Proc Natl Acad Sci USA 86:9692-9696; Holtorf et al.(1995) Plant Mol Biol 29:637-649), the mas, Mac or DoubleMac promoters(U.S. Pat. No. 5,106,739; Comai et al. (1990) Plant Mol Biol15:373-381), the ubiquitin promoter (Holtorf et al. (1995) Plant MolBiol 29:637-649), Rubisco small subunit (SSU) promoter (U.S. Pat. No.4,962,028), the legumin B promoter (GenBank Acc. No. X03677), thepromoter of the nopaline synthase (NOS) from Agrobacterium, the TR dualpromoter, the octopine synthase (OCS) promoter from Agrobacterium, theSmas promoter, the cinnamyl alcohol dehydrogenase promoter (U.S. Pat.No. 5,683,439), the promoters of the vacuolar ATPase subunits, the pEMUpromoter (Last et al. (1991) Theor. Appl. Genet. 81, 581-588); the MASpromoter (Velten et al. (1984) EMBO J. 3(12): 2723-2730), the maize H3histone promoter (Lepetit et al. (1992) Mol. Gen. Genet. 231: 276-285;Atanassova et al. (1992) Plant J 2(3): 291-300), □-conglycinin promoter,the phaseolin promoter, the ADH promoter, and heatshock promoters, thenitrilase promoter from Arabidopsis thaliana (WO 03/008596; GenBank Acc.No.: U38846, nucleotides 3,862 to 5,325 or else 5342), promoter of aproline-rich protein from wheat (WO 91/13991), the promoter of the Pisumsativum ptxA gene, and other transcription initiation regions fromvarious plant genes known to those of skill in the art.

However, it has to be noted that because of the high efficiency of thechimeric RNA of the invention, the method of the current invention doesnot rely on the presence of strong promoter regions to drive thetranscriptional production of the chimeric RNA. In other words, a wholerange of promoters, particularly plant expressible promoters, isavailable to direct the transcription.

3.1.3.1.1.2 Tissue-Specific Promoters

Alternatively promoters can be employed which regulate expression inonly one or some tissues or organs, such as leaves, roots, fruit, seeds,anthers, ovaries, pollen, meristem, stems or flowers, or parts thereof.For example, the tissue-specific ES promoter from tomato is particularlyuseful for directing gene expression so that a desired gene product islocated in fruits (see, e.g., Lincoln et al. (1988) Proc Natl Acad SciUSA 84:2793-2797; Deikman et al. (1988) EMBO J 7:3315-3320; Deikman etal. (1992) Plant Physiol 100:2013-2017). Suitable seed specificpromoters include those derived from the following genes: MAC1 frommaize (Sheridan et al. (1996) Genetics 142:1009-1020), Cat3 from maize(GenBank No. L05934), the gene encoding oleosin 18 kD from maize(GenBank No. J05212) viviparous-1 from Arabidopsis (Genbank Acc.-No.U93215), the gene encoding oleosin from Arabidopsis (Genbank Acc.-No.Z17657), Atmycl from Arabidopsis (Urao et al. (1996) Plant Mol Biol32:571-576), the 2S seed storage protein gene family from Arabidopsis(Conceicao et al. (1994) Plant 5:493-505) the gene encoding oleosin 20kD from Brassica napus (GenBank No. M63985), napin from Brassica napus(GenBank No. J02798, Joseffson et al. (1987) J Biol Chem262:12196-12201), the napin gene family (e.g., from Brassica napus;Sjodahl et al. (1995) Planta 197:264-271), U.S. Pat. No. 5,608,152;Stalberg et al. (1996) Planta 199:515-519), the gene encoding the 2Sstorage protein from Brassica napus (Dasgupta et al. (1993) Gene 133:301-302), the genes encoding oleosin A (Genbank Acc.-No. U09118) andoleosin B (Genbank No. U09119) from soybean, the gene encoding lowmolecular weight sulphur rich protein from soybean (Choi et al. (1995)Mol Gen Genet 246:266-268), the phaseolin gene (U.S. Pat. No. 5,504,200,Bustos et al. (1989) Plant Cell 1(9):839-53; Murai et al. (1983) Science23: 476-482; SenguptaGopalan et al. (1985) Proc. Nat'l Acad. Sci. USA82: 3320-3324 (1985)), the 2S albumin gene, the legumin gene (Shirsat etal. (1989) Mol Gen Genet 215(2):326-331), the USP (unknown seed protein)gene, the sucrose binding protein gene (WO 00/26388), the legumin B4gene (LeB4; Fiedler et al. (1995) Biotechnology (NY) 13(10):1090-1093),Bäumlein et al. (1992) Plant J 2(2):233-239; Bäumlein et al. (1991a) MolGen Genet 225(3):459-467; Bäumlein et al. (1991b) Mol Gen Genet225:121-128), the Arabidopsis oleosin gene (WO 98/45461), the BrassicaBce4 gene (WO 91/13980), genes encoding the “high-molecular-weightglutenin” (HMWG), gliadin, branching enzyme, ADP-glucose pyrophosphatase(AGPase) or starch synthase. Furthermore preferred promoters are thosewhich enable seed-specific expression in monocots such as maize, barley,wheat, rye, rice and the like. Promoters which may advantageously beemployed are the promoter of the Ipt2 or Ipt1 gene (WO 95/15389, WO95/23230) or the promoters described in WO 99/16890 (promoters of thehordein gene, the glutelin gene, the oryzin gene, the prolamine gene,the gliadin gene, the zein gene, the kasirin gene or the secalin gene).Further preferred are a leaf-specific and light-induced promoter such asthat from cab or Rubisco (Timko et al. (1985) Nature 318: 579-582;Simpson et al. (1985) EMBO J 4:2723-2729); an anther-specific promotersuch as that from LAT52 (Twell et al. (1989) Mol Gen Genet 217:240-245);a pollen-specific promoter such as that from ZmI3 (Guerrero et al.(1993) Mol Gen Genet 224:161-168); and a microspore-preferred promotersuch as that from apg (Twell et al. (1983) Sex. Plant Reprod.6:217-224). Further suitable promoters are, for example, specificpromoters for tubers, storage roots or roots such as, for example, theclass I patatin promoter (B33), the potato cathepsin D inhibitorpromoter, the starch synthase (GBSS1) promoter or the sporamin promoter,and fruit-specific promoters such as, for example, the tomatofruit-specific promoter (EP-A 409 625). Promoters which are furthermoresuitable are those which ensure leaf-specific expression. Promoterswhich may be mentioned are the potato cytosolic FBPase promoter (WO98/18940), the Rubisco (ribulose-1,5-bisphosphate carboxylase) SSU(small subunit) promoter or the potato ST-LSI promoter (Stockhaus et al.(1989) EMBO J 8(9):2445-2451). Other preferred promoters are those whichgovern expression in seeds and plant embryos. Further suitable promotersare, for example, fruit-maturation-specific promoters such as, forexample, the tomato fruit-maturation-specific promoter (WO 94/21794),flower-specific promoters such as, for example, the phytoene synthasepromoter (WO 92/16635) or the promoter of the P1-rr gene (WO 98/22593)or another node-specific promoter as described in EP-A 249676 may beused advantageously. The promoter may also be a pithspecific promoter,such as the promoter isolated from a plant TrpA gene as described in WO93/07278.

3.1.3.1.1.3 Chemically Inducible Promoters

An expression constructs may also contain a chemically induciblepromoter (review article: Gatz et al. (1997) Annu Rev Plant PhysiolPlant Mol Biol 48:89-108), by means of which the expression of thenucleic acid sequence encoding the chimeric RNA of the invention in theplant can be controlled at a particular point in time. Such promoterssuch as, for example, a salicylic acid-inducible promoter (WO 95/19443),a benzenesulfonamide-inducible promoter (EP 0 388 186), atetracycline-inducible promoter (Gatz et al. (1991) Mol Gen Genetics227:229-237), an abscisic acid-inducible promoter EP 0 335 528) or anethanol-cyclohexanone-inducible promoter (WO 93/21334) can likewise beused. Also suitable is the promoter of the glutathione-S transferaseisoform II gene (GSTII-27), which can be activated by exogenouslyapplied safeners such as, for example, N,N-diallyl-2,2-dichloroacetamide(WO 93/01294) and which is operable in a large number of tissues of bothmonocotyledonous and dicotyledonous. Further exemplary induciblepromoters that can be utilized in the instant invention include thatfrom the ACE1 system which responds to copper (Mett et al. PNAS 90:4567-4571 (1993)); or the In2 promoter from maize which responds tobenzenesulfonamide herbicide safeners (Hershey et al. (1991) Mol GenGenetics 227:229-237; Gatz et al. (1994) Mol Gen Genetics 243:32-38). Apromoter that responds to an inducing agent to which plants do notnormally respond can be utilized. An exemplary inducible promoter is theinducible promoter from a steroid hormone gene, the transcriptionalactivity of which is induced by a glucocorticosteroid hormone (Schena etal. (1991) Proc Nat'l Acad Sci USA 88:10421). Other preferred promotersare promoters induced by biotic or abiotic stress, such as, for example,the pathogen-inducible promoter of the PRP1 gene (Ward et al. (1993)Plant Mol Biol 22:361-366), the tomato heat-inducible hsp80 promoter(U.S. Pat. No. 5,187,267), the potato chill-inducible alpha-amylasepromoter (WO 96/12814) or the wound-induced pinII promoter (EP-A1 0 375091).

3.1.3.1.1.4 Stress- or Pathogen-Inducible Promoters

One can use a promoter that directs expression environmental control.Examples of environmental conditions that may affect transcription byinducible promoters include biotic or abiotic stress factors or otherenvironmental conditions, for example, pathogen attack, anaerobicconditions, ethylene or the presence of light.

Promoters inducible by biotic or abiotic stress include but are notlimited to the pathogen-inducible promoter of the PRP1 gene (Ward et al.(1993) Plant Mol Biol 22:361-366), the heat-inducible hsp70 or hsp80promoter from tomato (U.S. Pat. No. 5,187,267), the chill-induciblealpha-amylase promoter from potato (WO 96/12814), the light-induciblePPDK promoter or the wounding-inducible pinII promoter (EP375091).Pathogen-inducible promoters comprise those of genes which are inducedas the result of attack by pathogens such as, for example, genes of PRproteins, SAR proteins, b-1,3-glucanase, chitinase and the like (forexample Redolfi et al. (1983) Neth J Plant Pathol 89:245-254; Uknes, etal. (1992) The Plant Cell 4:645-656; Van Loon (1985) Plant Mol Viral4:111-116; Marineau et al. (1987) Plant Mol Biol 9:335-342; Matton etal. (1987) Molecular Plant-Microbe Interactions 2:325-342; Somssich etal. (1986) Proc Natl Acad Sci USA 83:2427-2430; Somssich et al. (1988)Mol Gen Genetics 2:93-98; Chen et al. (1996) Plant J 10:955-966; Zhangand Sing (1994) Proc Natl Acad Sci USA 91:2507-2511; Warner, et al.(1993) Plant J 3:191-201; Siebertz et al. (1989) Plant Cell1:961-968(1989)). Also comprised are wounding-inducible promoters suchas that of the pinII gene (Ryan (1990) Ann Rev Phytopath 28:425-449;Duan et al. (1996) Nat Biotech 14:494-498), of the wun1 and wun2 gene(U.S. Pat. No. 5,428,148), of the win1 and win2 gene (Stanford et al.(1989) Mol Gen Genet 215:200-208), of systemin (McGurl et al. (1992)Science 225:1570-1573), of the WIP1 gene (Rohmeier et al. (1993) PlantMol Biol 22:783-792; Eckelkamp et al. (1993) FEBS Letters 323:73-76), ofthe MPI gene (Corderok et al. (1994) The Plant J 6(2):141-150) and thelike.

3.1.3.1.1.5 Development-Dependent Promoters

Further suitable promoters are, for example, fruit-maturation-specificpromoters, such as, for example, the fruit-maturation-specific promoterfrom tomato (WO 94/21794, EP 409 625). Development-dependent promotersinclude partly the tissue-specific promoters described above sinceindividual tissues are, naturally, formed as a function of thedevelopment. A development-regulated promoter is, inter alia, described(Baerson and Lamppa (1993) Plant Mol Biol 22(2):255-67).

3.1.3.1.1.6 Other Suitable Promoter and Promoter Elements

Promoters may also encompass further promoters, promoter elements orminimal promoters capable of modifying the expression-governingcharacteristics. Thus, for example, the tissue-specific expression maytake place in addition as a function of certain stress factors, owing togenetic control sequences. Such elements are, for example, described forwater stress, abscisic acid (Lam and Chua (1991) J Biol Chem266(26):17131-17135) and heat stress (Schoffl et al. (1989) Molecular &General Genetics 217(2-3):246-53).

3.1.3.1.2 Other Genetic Control Elements

Genetic control sequences are furthermore to be understood as thosepermitting removal of the inserted sequences from the genome. Methodsbased on the cre/lox (Dale and Ow (1991) Proc Nat'l Acad Sci USA88:10558-10562; Sauer (1998) Methods 14(4):381-92; Odell et al. (1990)Mol Gen Genet 223:369-378), FLP/FRT (Lysnik et al. (1993) NAR21:969-975), or Ac/Ds system (Lawson et al. (1994) Mol Gen Genet245:608-615; Wader et al. (1987) in TOMATO TECHNOLOGY 189-198 (Alan R.Liss, Inc.); U.S. Pat. No. 5,225,341; Baker et al. (1987) EMBO J 6:1547-1554) permit a—if appropriate tissue-specific and/orinducible—removal of a specific DNA sequence from the genome of the hostorganism. Control sequences may in this context mean the specificflanking sequences (e.g., lox sequences), which later allow removal(e.g., by means of cre recombinase).

3.1.3.1.2.1 Transcriptional Terminators

A variety of transcriptional terminators are available for use inexpression constructs. These are responsible for the termination oftranscription beyond the transgene and its correct polyadenylation.Appropriate transcriptional terminators are those that are known tofunction in plants and include the CaMV 35S terminator, the tmlterminator, the OCS (octopin synthase) terminator and the NOS (nopalinsynthase) terminator and the pea rbcS E9 terminator. These can be usedin both monocotyledons and dicotyledons.

3.1.3.1.2.2 Sequences for the Enhancement or Regulation of Expression

Genetic control sequences furthermore also comprise the 5′-untranslatedregions, introns or noncoding 3′ region of genes, such as, for example,the actin-1 intron, or the Adh1-S introns 1, 2 and 6 (general reference:The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer,New York (1994)). It has been demonstrated that they can play asignificant role in the regulation of gene expression and have beenshown to enhance expression, particularly in monocotyledonous cells.Thus, it has been demonstrated that 5′-untranslated sequences canenhance the transient expression of heterologous genes. An example whichmay be mentioned of such translation enhancers is the tobacco mosaicvirus 5′ leader sequence (Gallie et al. (1987) Nucl Acids Res15:8693-8711) and the like. They can furthermore promote tissuespecificity (Rouster J et al. (1998) Plant J 15:435-440).

3.1.3.2. Construction of Plant Transformation Vectors

The expression construct for expression of the chimeric RNA molecule ofthe invention is preferably comprised in an expression vector. Numeroustransformation vectors for plant transformation are known to the personskilled in the plant transformation arts. The selection of vector willdepend upon the preferred transformation technique and the targetspecies for transformation.

3.1.3.2.1 Vector Elements

Expression constructs and the vectors derived therefrom may comprisefurther functional elements. The term functional element is to beunderstood in the broad sense and means all those elements, which havean effect on the generation, multiplication or function of theexpression constructs, vectors or transgenic organisms according to theinvention. The following may be mentioned by way of example, but not bylimitation:

3.1.3.2.1.1. Selectable Marker Genes

Selectable marker genes are useful to select and separate successfullytransformed cells. Preferably, within the method of the invention onemarker may be employed for selection in a prokaryotic host, whileanother marker may be employed for selection in a eukaryotic host,particularly the plant species host. The marker may confer resistanceagainst a biocide, such as antibiotics, toxins, heavy metals, or thelike, or may function by complementation, imparting prototrophy to anauxotrophic host. Preferred selectable marker genes for plants mayinclude but are not be limited to the following:

3.1.3.2.1.1.1. Negative Selection Markers

Negative selection markers confer a resistance to a biocidal compoundsuch as a metabolic inhibitor (e.g., 2-deoxyglucose-6-phosphate, WO98/45456), antibiotics (e.g., kanamycin, G 418, bleomycin or hygromycin)or herbicides (e.g., phosphinothricin or glyphosate). Especiallypreferred negative selection markers are those which confer resistanceto herbicides. These markers can be used—beside their function as amarker—to confer a herbicide resistance trait to the resulting plant.Examples, which may be mentioned, are:

-   -   Phosphinothricin acetyltransferases (PAT; also named Bialophos        resistance; bar; de Block et al. (1987) EMBO J 6:2513-2518; EP 0        333 033; U.S. Pat. No. 4,975,374)    -   5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; U.S. Pat.        No. 5,633,435) or glyphosate oxidoreductase gene (U.S. Pat. No.        5,463,175) conferring resistance to Glyphosate (Nphosphonomethyl        glycine) (Shah et al. (1986) Science 233: 478)    -   Glyphosate degrading enzymes (Glyphosate oxidoreductase; gox),    -   Dalapon inactivating dehalogenases (deh)    -   Sulfonylurea- and imidazolinone-inactivating acetolactate        synthases (for example mutated ALS variants with, for example,        the S4 and/or Hra mutation    -   Bromoxynil degrading nitrilases (bxn)    -   Kanamycin- or. G418-resistance genes (NPTII; NPTI) coding e.g.,        for neomycin phosphotransferases (Fraley et al. (1983) Proc Natl        Acad Sci USA 80:4803), which expresses an enzyme conferring        resistance to the antibiotic kanamycin and the related        antibiotics neomycin, paromomycin, gentamicin, and G418,    -   2-Deoxyglucose-6-phosphate phosphatase (DOGR1-Gene product; WO        98/45456; EP 0 807 836) conferring resistance against        2-desoxyglucose (Randez-Gil et al. (1995) Yeast 11:1233-1240)    -   Hygromycin phosphotransferase (HPT), which mediates resistance        to hygromycin (Vanden Elzen et al. (1985) Plant Mol Biol.        5:299).    -   Dihydrofolate reductase (Eichholtz et al. (1987) Somatic Cell        and Molecular Genetics 13, 67-76)

Additional negative selectable marker genes of bacterial origin thatconfer resistance to antibiotics include the aadA gene, which confersresistance to the antibiotic spectinomycin, gentamycin acetyltransferase, streptomycin phosphotransferase (SPT),aminoglycoside-3-adenyl transferase and the bleomycin resistancedeterminant (Svab et al. (1990) Plant Mol. Biol. 14:197; Jones et al.(1987) Mol. Gen. Genet. 210:86; Hille et al. (1986) Plant Mol. Biol.7:171 (1986); Hayford et al. (1988) Plant Physiol. 86:1216).

Especially preferred are negative selection markers which conferresistance against the toxic effects imposed by D-amino acids like e.g.,D-alanine and D-serine (WO 03/060133; Erikson et al. Nat Biotechnol.22(4):455-8 (2004)). Especially preferred as negative selection markerin this contest are the daol gene (EC: 1.4. 3.3: GenBank Acc.-No.:U60066) from the yeast Rhodotorula gracilis (Rhodosporidium toruloides)and the E. coli gene dsdA (D-serine dehydratase (D-serine deaminase)[EC: 4.3. 1.18; GenBank Acc.-No.: J01603). Depending on the employedD-amino acid the D-amino acid oxidase markers can be employed as dualfunction marker offering negative selection (e.g., when combined withfor example D-alanine or D-serine) or counter selection (e.g., whencombined with D-leucine or D-isoleucine).

3.1.3.2.1.1.2. Positive Selection Marker

Positive selection markers are conferring a growth advantage to atransformed plant in comparison with a non-transformed one. Genes likeisopentenyltransferase from Agrobacterium tumefaciens (strain:PO22;Genbank Acc.-No.: AB025109) may—as a key enzyme of the cytokininbiosynthesis—facilitate regeneration of transformed plants (e.g., byselection on cytokinin-free medium). Corresponding selection methods aredescribed (Ebinuma et al. (2000a) Proc Natl Acad Sci USA 94:2117-2121;Ebinuma et al. (2000b) Selection of Marker-free transgenic plants usingthe oncogenes (ipt, rol A, B, C) of Agrobacterium as selectable markers,In Molecular Biology of Woody Plants. Kluwer Academic Publishers).Additional positive selection markers, which confer a growth advantageto a transformed plant in comparison with a non-transformed one, aredescribed e.g., in EP-A 0 601 092. Growth stimulation selection markersmay include (but shall not be limited to) □-Glucuronidase (incombination with e.g., cytokinin glucuronide), mannose-6-phosphateisomerase (in combination with mannose), UDP-galactose-4-epimerase (incombination with e.g., galactose), wherein mannose-6-phosphate isomerasein combination with mannose is especially preferred.

3.1.3.2.1.1.3. Counter Selection Marker

Counter selection markers are especially suitable to select organismswith defined deleted sequences comprising said marker (Koprek et al.(1999) Plant J 19(6): 719-726). Examples for counter selection markercomprise thymidine kinases (TK), cytosine deaminases (Gleave et al.(1999) Plant Mol Biol. 40(2):223-35; Perera et al. (1993) Plant Mol.Biol 23(4): 793-799; Stougaard (1993) Plant J 3:755-761), cytochrom P450proteins (Koprek et al. (1999) Plant J 19(6): 719-726), haloalkandehalogenases (Naested (1999) Plant J 18:571-576), iaaH gene products(Sundaresan et al. (1995) Gene Develop 9: 1797-1810), cytosine deaminasecodA (Schlaman and Hooykaas (1997) Plant J 11:1377-1385), or tms2 geneproducts (Fedoroff and Smith (1993) Plant J 3:273-289).

3.1.3.2.1.2. Reporter Genes

Reporter genes encode readily quantifiable proteins and, via their coloror enzyme activity, make possible an assessment of the transformationefficacy, the site of expression or the time of expression. Veryespecially preferred in this context are genes encoding reporterproteins (Schenborn and Groskreutz (1999) Mol Biotechnol 13(1):29-44)such as the green fluorescent protein (GFP) (Haseloff et al. (1997) ProcNatl Acad Sci USA 94(6):2122-2127; Sheen et al. (1995) Plant J8(5):777-784; Reichel et al. (1996) Proc Natl Acad Sci USA93(12):5888-5893; Chui et al. (1996) Curr Biol 6:325-330; Leffel et al.(1997) Biotechniques. 23(5):912-8; Tian et al. (1997) Plant Cell Rep16:267-271; WO 97/41228), chloramphenicol transferase, a luciferase(Millar et al. (1992) Plant Mol Biol Rep 10:324-414; Ow et al. (1986)Science 234:856-859), the aequorin gene (Prasher et al. (1985) BiochemBiophys Res Commun 126(3):1259-1268), β-galactosidase, R locus gene(encoding a protein which regulates the production of anthocyaninpigments (red coloring) in plant tissue and thus makes possible thedirect analysis of the promoter activity without addition of furtherauxiliary substances or chromogenic substrates (Dellaporta et al. (1988)In: Chromosome Structure and Function: Impact of New Concepts, 18thStadler Genetics Symposium, 11:263-282; Ludwig et al. (1990) Science247:449), with β-glucuronidase (GUS) being very especially preferred(Jefferson (1987b) Plant Mol. Bio. Rep., 5:387-405; Jefferson et al.(1987) EMBO J 6:3901-3907). β-glucuronidase (GUS) expression is detectedby a blue color on incubation of the tissue with5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid, bacterial luciferase(LUX) expression is detected by light emission; firefly luciferase (LUC)expression is detected by light emission after incubation withluciferin; and galactosidase expression is detected by a bright bluecolor after the tissue was stained with5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside. Reporter genes mayalso be used as scorable markers as alternatives to antibioticresistance markers. Such markers are used to detect the presence or tomeasure the level of expression of the transferred gene. The use ofscorable markers in plants to identify or tag genetically modified cellsworks well only when efficiency of modification of the cell is high.

3.1.3.2.1.3. Origins of Replication.

Origins of replication which ensure amplification of the expressionconstructs or vectors ac-cording to the invention in, for example, E.coli. Examples which may be mentioned are ORI (origin of DNAreplication), the pBR322 ori or the P15A ori (Maniatis T, Fritsch E Fand Sambrook J (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor (N.Y.)). Additionalexamples for replication systems functional in E. coli, are ColE1,pSC101, pACYC184, or the like. In addition to or in place of the E. colireplication system, a broad host range replication system may beemployed, such as the replication systems of the P-1 Incompatibilityplasmids; e.g., pRK290. These plasmids are particularly effective witharmed and disarmed Ti-plasmids for transfer of T-DNA to the plant host.

3.1.3.2.1.4. Elements, which are necessary for Agrobacterium-mediatedtransformation, such as, for example, the right and/or—optionally—leftborder of the T-DNA or the vir region.

3.1.3.2.1.5. Multiple cloning sites (MCS) to enable and facilitate theinsertion of one or more nucleic acid sequences.

3.1.3.2.2 Vectors for Plant Transformation

3.1.3.2.2.1 Vectors Suitable for Agrobacterium Transformation

If Agrobacteria are used, the expression construct is to be integratedinto specific plasmids vectors, either into a shuttle, or intermediate,vector or into a binary vector. If a Ti or Ri plasmid is to be used forthe transformation, at least the right border, but in most cases theright and the left border, of the Ti or Ri plasmid T-DNA is flanking theregion with the expression construct to be introduced into the plantgenome. It is preferred to use binary vectors for the Agrobacteriumtransformation. Binary vectors are capable of replicating both in E.coli and in Agrobacterium. They preferably comprise a selection markergene and a linker or polylinker flanked by the right and—optionally—leftT-DNA border sequence. They can be transformed directly intoAgrobacterium (Holsters et al. (1978) Mol Gen Genet 163:181-187). Aselection marker gene may be included in the vector which permits aselection of transformed Agrobacteria (e.g., the nptIII gene). TheAgrobacterium, which acts as host organism in this case, should alreadycomprise a disarmed (i.e., non-oncogenic) plasmid with the vir region.This region is required for transferring the T-DNA to the plant cell.The use of T-DNA for the transformation of plant cells has been studiedand described extensively (EP 120 516; Hoekema, In: The Binary PlantVector System, Offsetdrukkerij Kanters B.V., Alblasserdam, Chapter V; Anet al. (1985) EMBO J 4:277-287). A variety of binary vectors are knownand available for transformation using Agrobacterium, such as, forexample, pBI101.2 or pBIN19 (Clontech Laboratories, Inc. USA; Bevan etal. (1984) Nucl Acids Res 12:8711), pBinAR, pPZP200 or pPTV.

3.1.3.2.2.2 Vectors Suitable for Non-Agrobacterium Transformation

Transformation without the use of Agrobacterium tumefaciens circumventsthe requirement for T-DNA sequences in the chosen transformation vectorand consequently vectors lacking these sequences can be utilized inaddition to vectors such as the ones described above which contain T-DNAsequences. Transformation techniques that do not rely on Agrobacteriuminclude transformation via particle bombardment, protoplast uptake (e.g.PEG and electroporation) and microinjection. The choice of vectordepends largely on the preferred selection for the species beingtransformed. Typical vectors suitable for non-Agrobacteriumtransformation include pCIB3064, pSOG19, and pSOG35. (See, for example,U.S. Pat. No. 5,639,949).

3.1.3.3. Transformation Techniques

3.1.3.3.1 General Techniques

Once an expression construct or expression vector of the invention hasbe established, it can be transformed into a plant cell. A variety ofmethods for introducing nucleic acid sequences (e.g., vectors) into thegenome of plants and for the regeneration of plants from plant tissuesor plant cells are known (Plant Molecular Biology and Biotechnology (CRCPress, Boca Raton, Fla.), chapter 6/7, pp. 71-119 (1993); White F F(1993) Vectors for Gene Transfer in Higher Plants; in: TransgenicPlants, vol. 1, Engineering and Utilization, Ed.: Kung and Wu R,Academic Press, 15-38; Jenes B et al. (1993) Techniques for GeneTransfer, in: Transgenic Plants, vol. 1, Engineering and Utilization,Ed.: Kung and R. Wu, Academic Press, pp. 128-143; Potrykus (1991) AnnuRev Plant Physiol Plant Molec Biol 42:205-225; Halford N G, Shewry P R(2000) Br Med Bull 56(1):62-73).

Transformation methods may include direct and indirect methods oftransformation. Suitable direct methods include polyethylene glycolinduced DNA uptake, liposome-mediated transformation (U.S. Pat. No.4,536,475), biolistic methods using the gene gun (“particlebombardment”; Fromm M E et al. (1990) Bio/Technology. 8(9):833-9;Gordon-Kamm et al. (1990) Plant Cell 2:603), electroporation, incubationof dry embryos in DNA-comprising solution, and microinjection. In thecase of these direct transformation methods, the plasmid used need notmeet any particular requirements. Simple plasmids, such as those of thepUC series, pBR322, M13mp series, pACYC184 and the like can be used. Ifintact plants are to be regenerated from the transformed cells, anadditional selectable marker gene is preferably located on the plasmid.The direct transformation techniques are equally suitable fordicotyledonous and monocotyledonous plants.

Transformation can also be carried out by bacterial infection by meansof Agrobacterium (for example EP 0 116 718), viral infection by means ofviral vectors (EP 0 067 553; U.S. Pat. No. 4,407,956; WO 95/34668; WO93/03161) or by means of pollen (EP 0 270 356; WO 85/01856; U.S. Pat.No. 4,684,611). Agrobacterium based transformation techniques(especially for dicotyledonous plants) are well known in the art. TheAgrobacterium strain (e.g., Agrobacterium tumefaciens or Agrobacteriumrhizogenes) comprises a plasmid (Ti or Ri plasmid) and a T-DNA elementwhich is transferred to the plant following infection withAgrobacterium. The T-DNA (transferred DNA) is integrated into the genomeof the plant cell. The T-DNA may be localized on the Ri- or Ti-plasmidor is separately comprised in a so-called binary vector. Methods for theAgrobacterium-mediated transformation are described, for example, inHorsch R B et al. (1985) Science 225:1229f. The Agrobacterium-mediatedtransformation is best suited to dicotyledonous plants but has also beadopted to monocotyledonous plants. The transformation of plants byAgrobacteria is described (White F F, Vectors for Gene Transfer inHigher Plants; in Transgenic Plants, Vol. 1, Engineering andUtilization, edited by S. D. Kung and R. Wu, Academic Press, 1993, pp.15-38; Jenes B et al. (1993) Techniques for Gene Transfer, in:Transgenic Plants, Vol. 1, Engineering and Utilization, edited by S. D.Kung and R. Wu, Academic Press, pp. 128-143; Potrykus (1991) Annu RevPlant Physiol Plant Molec Biol 42:205-225).

Transformation may result in transient or stable transformation andexpression. Although a nucleotide sequence of the present invention canbe inserted into any plant and plant cell falling within these broadclasses (as specified above in the DEFINITION section), it isparticularly useful in crop plant cells.

Various tissues are suitable as starting material (explant) for theAgrobacterium-mediated transformation process including but not limitedto callus (U.S. Pat. No. 5,591,616; EP-A1 604 662), immature embryos(EP-A1 672 752), pollen (U.S. Pat. No. 54,929,300), shoot apex (U.S.Pat. No. 5,164,310), or in planta transformation (U.S. Pat. No.5,994,624). The method and material described herein can be combinedwith virtually all Agrobacterium mediated transformation methods knownin the art. Preferred combinations include—but are not limited—to thefollowing starting materials and methods:

TABLE 1 Plant Transformation Methods Variety Material/CitationMonocotyledonous Immature embryos (EP-A1 672 752) plants: Callus (EP-A1604 662) Embryogenic callus (U.S. Pat. No. 6,074,877) Inflorescence(U.S. Pat. No. 6,037,522) Flower (in planta) (WO 01/12828) Banana U.S.Pat. No. 5,792,935; EP-A1 731 632; U.S. Pat. No. 6,133,035 Barley WO99/04618 Maize U.S. Pat. No. 5,177,010; U.S. Pat. No. 5,987,840Pineapple U.S. Pat. No. 5,952,543; WO 01/33943 Rice EP-A1 897 013; U.S.Pat. No. 6,215,051; WO 01/12828 Wheat AU-B 738 153; EP-A1 856 060 BeansU.S. Pat. No. 5,169,770; EP-A1 397 687 Brassica U.S. Pat. No. 5,188,958;EP-A1 270 615; EP-A1 1,009,845 Cacao U.S. Pat. No. 6,150,587 Citrus U.S.Pat. No. 6,103,955 Coffee AU 729 635 Cotton U.S. Pat. No. 5,004,863;EP-A1 270 355; U.S. Pat. No. 5,846,797; EP-A1 1,183,377; EP-A11,050,334; EP-A1 1,197,579; EP-A1 1,159,436 Pollen transformation (U.S.Pat. No. 5,929,300) In planta transformation (U.S. Pat. No. 5,994,624)Pea U.S. Pat. No. 5,286,635 Pepper U.S. Pat. No. 5,262,316 Poplar U.S.Pat. No. 4,795,855 Soybean cotyledonary node of germinated soybeanseedlings shoot apex (U.S. Pat. No. 5,164,310) axillary meristematictissue of primary, or higher leaf node of about 7 days germinatedsoybean seedlings organogenic callus cultures dehydrated embryo axesU.S. Pat. No. 5,376,543; EP-A1 397 687; U.S. Pat. No. 5,416,011; U.S.Pat. No. 5,968,830; U.S. Pat. No. 5,563,055; U.S. Pat. No. 5,959,179;EP-A1 652 965; EP-A1 1,141,346 Sugarbeet EP-A1 517 833; WO 01/42480Tomato U.S. Pat. No. 5,565,347

3.1.3.3.2. Plastid Transformation

In another preferred embodiment, a nucleotide sequence of the presentinvention (preferably an expression construct for the chimeric RNAmolecule of the invention) is directly transformed into the plastidgenome. Plastid expression, in which genes are inserted by homologousrecombination into the several thousand copies of the circular plastidgenome present in each plant cell, takes advantage of the enormous copynumber advantage over nuclear-expressed genes to permit high expressionlevels. In a preferred embodiment, the nucleotide sequence is insertedinto a plastid targeting vector and transformed into the plastid genomeof a desired plant host. Plants homoplasmic for plastid genomescontaining the nucleotide sequence are obtained, and are preferentiallycapable of high expression of the nucleotide sequence.

Plastid transformation technology is for example extensively describedin U.S. Pat. Nos. 5,451,513, 5,545,817, 5,545,818, and 5,877,462 in PCTapplication no. WO 95/16783 and WO 97/32977, and in McBride et al.(1994) Proc. Natl. Acad. Sci. USA 91, 7301-7305, all incorporated hereinby reference in their entirety. The basic technique for plastidtransformation involves introducing regions of cloned plastid DNAflanking a selectable marker together with the nucleotide sequence intoa suitable target tissue, e.g., using biolistic or protoplasttransformation (e.g., calcium chloride or PEG mediated transformation).The 1 to 1.5 kb flanking regions, termed targeting sequences, facilitatehomologous recombination with the plastid genome and thus allow thereplacement or modification of specific regions of the plastome.Initially, point mutations in the chloroplast 16S rRNA and rps12 genesconferring resistance to spectinomycin and/or streptomycin are utilizedas selectable markers for transformation (Svab et al. (1990) Proc. Natl.Acad. Sci. USA 87, 8526-8530; Staub et al. (1992) Plant Cell 4, 39-45).The presence of cloning sites between these markers allowed creation ofa plastid targeting vector for introduction of foreign genes (Staub etal. (1993) EMBO J. 12, 601-606). Substantial increases in transformationfrequency are obtained by replacement of the recessive rRNA or r-proteinantibiotic resistance genes with a dominant selectable marker, thebacterial aadA gene encoding the spectinomycin-detoxifying enzymeaminoglycoside-3′-adenyltransferase (Svab et al. (1993) Proc. Natl.Acad. Sc. USA 90, 913-917). Other selectable markers useful for plastidtransformation are known in the art and encompassed within the scope ofthe invention.

For using the methods according to the invention, the skilled worker hasavailable well-known tools, such as expression vectors with promoterswhich are suitable for plants, and methods for the transformation andregeneration of plants.

3.1.3.4. Selection and Regeneration Techniques

To select cells which have successfully undergone transformation, it ispreferred to introduce a selectable marker which confers, to the cellswhich have successfully undergone transformation, a resistance to abiocide (for example a herbicide), a metabolism inhibitor such as2-deoxyglucose-6-phosphate (WO 98/45456) or an antibiotic. The selectionmarker permits the transformed cells to be selected from untransformedcells (McCormick et al. (1986) Plant Cell Reports 5:81-84). Suitableselection markers are described above.

Transgenic plants can be regenerated in the known manner from thetransformed cells. The resulting plantlets can be planted and grown inthe customary manner. Preferably, two or more generations should becultured to ensure that the genomic integration is stable andhereditary. Suitable methods are described (Fennell et al. (1992) PlantCell Rep. 11: 567-570; Stoeger et al (1995) Plant Cell Rep. 14:273-278;Jahne et al. (1994) Theor Appl Genet 89:525-533).

3.2 Pharmaceutical (Therapeutic or Prophylactic) Compositions andMethods

The specificity of compounds, compositions and methods of the inventioncan also be harnessed by those of skill in the art for therapeutic orprophylactic uses and are suitable for the preparation ofpharmaceuticals for the treatment of human and animal diseases and forthe production of pharmaceuticals.

Thus, the invention further provides a method for treating or preventinga disease or infection in an animal or human being, preferably a mammal.Yet another embodiment of the invention relates to a pharmaceuticallypreparation comprising at least one chimeric RNA of the invention.Preferably, said preparation gives rise to

-   i) at least one protein which has a therapeutic or prophylactic    effect on the target organism (preferably an animal or human) or-   ii) at least one functional RNA molecule, which to attenuates    expression of at least one disease-related target gene.

Yet another embodiment relates a chimeric RNA of the invention, anexpression construct or expression vector for its expression, or anorganism (preferably a non-human organism) comprising said chimeric RNAmolecule for use as a pharmaceutical, preferably for the treatment ofone or more human or animal diseases. Yet another embodiment relates tothe use of a chimeric RNA of the invention, an expression construct orexpression vector for its expression, or a non-human organism comprisingsaid chimeric RNA molecule for the preparation of a pharmaceutical,preferably for the treatment of one or more human or animal diseases.

The chimeric RNA of the invention (or a expression construct or vectorfor its expression) is administered to animal or human being (e.g., themammal) in a therapeutically or prophylactically effective amount (e.g.,an amount sufficient to attenuate expression of a target gene, theexpression of which is associated with the disease or infection; or—incase of protein expression—an amount suitable to bring about the effectassociated with the therapeutic protein). In case of disease genesuppression, the expression of the target gene (or alternatively theactivity of the target protein expressed therefrom) is inhibited by atleast about 10%, preferably by at least about 30%, more preferably by atleast 50% or more.

A variety of disorders can be treated, including infections byheterologous pathogenic organisms, either extracellular or intracellularpathogens. Additionally, the compositions of this invention are usefulin preventing infection with a pathogen, or preventing the occurrence ofdisorders caused by reactivation of a latent pathogen. Thesecompositions are also useful for the treatment of pathogenically-inducedcancers. The composition and methods of the invention are especiallysuitable to treat viral diseases (i.e., HIV, Hepatitis C). Thisespecially applies for gene silencing approaches.

Thus, the methods of the present invention employ a gene therapyconstruct comprising a nucleic acid molecule that encodes a polypeptidehaving a therapeutic biological activity (also referred to herein as a“therapeutic polypeptide”), including but not limited toimmunostimulatory molecules, tumor suppressor gene products/antigens,antimetabolites, suicide gene products, and anti-angiogenic factors. SeeMackensen et al. (1997) Cytokine Growth Factor Rev 8(2):119-128; Walther& Stein (1999) Mol Biotechnol 13(1):21-28; Kirk & Mule (2000) Hum GeneTher 11(6):797-806; and references cited therein.

Furthermore other (not pathogen related) disorders and diseases can betreated. Examples of diseases that can be treated by oligonucleotidecompositions include: cancer, retinopathies, autoimmune diseases,inflammatory diseases (i.e., ICAM-1 related disorders, Psoriasis,Ulcerative Colitus, Crohn's disease), cardiovascular diseases (such ashypertension), diseases of the central or peripheral nervous system suchas Alzheimer's disease, Parkinson's disease or multiple sclerosis, andautosomal dominant genetic disease such as Huntington's chorea (Forexample, see U.S. Pat. No. 6,506,559; US 2002/0,173,478 A1; US2002/0,086,356 A1; Shuey, et al., “RNAi: gene-silencing in therapeuticintervention.” Drug Discov. Today 2002 Oct. 15; 7(20):1040-6; Aoki, etal., “Clin. Exp. Pharmacol. Physiol. 2003 January; 30(1-2):96-102;Cioca, et al., “RNA interference is a functional pathway withtherapeutic potential in human myeloid leukemia cell lines. Cancer GeneTher. 2003 February; 10(2):125-33). There are numerous medicalconditions for which gene silencing therapy is reported to be suitable(see, e.g., U.S. Pat. No. 5,830,653) as well as respiratory syncytialvirus infection (WO 95/22,553) influenza virus (WO 94/23,028), andmalignancies (WO 94/08,003). Other examples of clinical uses ofantisense sequences are reviewed, e.g., in Glaser. 1996. GeneticEngineering News 16:1. Exemplary targets for cleavage byoligonucleotides include, e.g., protein kinase Ca, ICAM-1, c-raf kinase,p53, c-myb, and the bcr/abl fusion gene found in chronic myelogenousleukemia. The method of the invention can further be used to reduce orprevent the rejection response to transplant tissue (e.g., by silencingMHC proteins). A chimeric RNA hat attenuates the expression of a gene inthe transplant tissue that can elicit an immune response in therecipient is administered to the transplant tissue.

Also, the method according to the invention makes possible the paralleltreatment of more than one disease, such as, for example, acardiovascular disease and a disease of the central nervous system,which is not generally possible when traditional approaches are used.Such approaches are advantageous especially in the case of multiplediseases as occur frequently with advanced age. An example which may bementioned is the parallel treatment of hypertension and, for example,Alzheimer's disease or senile dementia.

The compounds and compositions of the invention can be utilized inpharmaceutical compositions by adding an effective amount of thecompound or composition to a suitable pharmaceutically acceptablediluent or carrier. Use of the oligomeric compounds and methods of theinvention may also be useful prophylactically.

3.2.1 Diseases and Disorders Preferred to be Treated by Compositions andMethods of the Invention

The method according to the invention is particularly suitable for thetreatment of the below mentioned diseases and disorders.

3.2.1.1 Pathogen Infections

Infection with pathogens, such as, for example, viral or bacterialdiseases, in which case the chimeric RNA (or the dsRNA derivedtherefrom) attenuates the expression of a bacterial or viral gene.Specifically some of the more desirable viruses to treat with thismethod include, without limitation, viruses of the species Retrovirus,Herpesvirus, Hepadenovirus, Poxvirus, Parvovirus, Papillomavirus, andPapovavirus, espcially HIV, HBV, HSV, CMV, HPV, HTLV and EBV. Thechimeric RNA used in this method provides to the cell (e.g., of anmammal) an at least partially double-stranded RNA molecule as describedabove, which is substantially identical to a target polynucleotide whichis a virus polynucleotide sequence necessary for replication and/orpathogenesis of the virus in an infected mammalian cell. Among suchtarget polynucleotide sequences are protein-encoding sequences forproteins necessary for the propagation of the virus, e.g., the HIV gag,env, gp41, and pol genes, the HPV6 L1 and E2 genes, the HPV11 L1 and E2genes, the HPV16 E6 and E7 genes, the HPV18 E6 and E7 genes, the HBVsurface antigens, the HBV core antigen, HBV reverse transcriptase, theHSV gD gene, the HSVvp16 gene, the HSV gC, gH, gL and gB genes, the HSVICPO, ICP4 and ICP6 genes, Varicella zoster gB, gC and GH genes, and theBCR-abl chromosomal sequences, and non-coding viral polynucleotidesequences which provide regulatory functions necessary for transfer ofthe infection from cell to cell, e.g., the HIV LTR, and other viralpromoter sequences, such as HSV vp16 promoter, HSV-ICPO promoter,HSV-ICP4, ICP6 and gD promoters, the HBV surface antigen promoter, theHBV pre-genomic promoter, among others. The composition (e.g., an dsRNAagent such as the chimeric RNA molecule of the invention) isadministered with an polynucleotide uptake enhancer or facilitator andan optional pharmaceutically acceptable carrier. The amount or dosagewhich is administered to the mammal is effective to reduce or inhibitthe function of the viral sequence in the cells of the mammal.

The method can be used to treat animals (e.g., mammals) already infectedwith a virus in order to shut down or inhibit a viral gene functionessential to virus replication and/or pathogenesis. In still anotherembodiment of this invention, the compositions described above can beemployed in a method to prevent viral infection (e.g., in a mammal).When the chimeric RNA of the invention is administered prior to exposureof the mammal to the virus, it is expected that the exogenous RNAmolecule remains in the mammal and work to inhibit any homologous viralsequence which presents itself to the mammal thereafter. Thus, thecompositions of the present invention may be used to inhibit or reducethe function of a viral polynucleotide sequence for vaccine use. Stillan analogous embodiment of the above “anti-viral” methods of theinvention includes a method for treatment or prophylaxis of a virallyinduced cancer in a mammal (such cancers include HPV E6/E7 virusinducedcervical carcinoma, and EBV induced cancers).

The compositions of this invention can also be employed for thetreatment or prophylaxis of infection by a non-viral pathogen, eitherintracellular or extracellular. As used herein, the term “intracellularpathogen” is meant to refer to a virus, bacteria, protozoan or otherpathogenic organism that, for at least part of its reproductive or lifecycle, exists within a host cell and therein produces or causes to beproduced, pathogenic proteins. Intracellular pathogens which infectcells which include a stage in the life cycle where they areintracellular pathogens include, without limitation, Listeria,Chlamydia, Leishmania, Brucella, Mycobacteria, Shigella, and as well asPlasmodia, e.g., the causative agent of malaria, P. falciparum.Extracellular pathogens are those which replicate and/or propagateoutside of the mammalian cell, e.g., Gonorrhoeae, and Borrellia, amongothers. According to this embodiment, such infection by an pathogen maybe treated or possibly prevented by administering to a mammaliansubject, either already infected or anticipating exposure to thepathogen, with a composition as described above with an optional secondagent that facilitates polynucleotide uptake in a cell, in apharmaceutically acceptable carrier. In this case, the RNA molecule ofthe composition has a polynucleotide sequence which is substantiallyidentical to a target polynucleotide sequence of the pathogen that isnecessary for replication and/or pathogenesis of the pathogen in aninfected mammal or mammalian cell. As above, the amount of thecomposition administered is an amount effective to reduce or inhibit thefunction of the pathogenic sequence in the mammal. The dosages, timing,routes of administration and the like are as described below.

Thus one embodiment of the invention related to a method for reducingthe susceptibility of host cells or host organisms to infection bypathogen, comprising introducing a chimeric RNA of the invention intosaid host cells or host organisms in an amount sufficient to attenuateexpression of one or more genes necessary for expression by saidpathogen. Preferably, the pathogen is a virus, a fungus or a nematode.Preferably, the host cell is a plant or an animal, preferably amammalian, more preferably a human cell.

One of skill in the art, given this disclosure can readily select viralfamilies and genera, or pathogens including prokaryotic and eukaryoticprotozoan pathogens as well as multicellular parasites, for whichtherapeutic or prophylactic compositions according to the presentinvention can be made. See, e.g., the tables of such pathogens ingeneral immunology texts and in U.S. Pat. No. 5,593,972, incorporated byreference herein.

3.2.1.2 Cancer

Treatment of cancer (for example solid tumors and/or leukemias) andinherited disorders. Among conditions particularly susceptible totreatment or prophylaxis according to this invention are thoseconditions which are characterized by the presence of an aberrantpolynucleotide sequence, the function of which is necessary to theinitiation or progression of the disorder, but can be inhibited withoutcausing harm or otherwise unduly adversely impacting the health of theorganism (e.g. the mammal). Mammalian cancers which are characterized bythe presence of abnormal and normal polynucleotide sequences (fordetails see, e.g., WO94/13793) include chronic myelogenous leukemia(CML) and acute lymphoblastic leukemia (ALL), where the abnormalsequence is a fusion of two normal genes, i.e., bcr-abl. In such cancersor diseases, such as CML, the afflicted mammal also possesses a normalcopy of the polynucleotide sequence or gene, and the differences betweenthe abnormal and normal sequences or genes are differences in nucleotidesequence. For example, for CML, the abnormal sequence is the bcr-ablfusion, while the normal sequence is bcr and abl. Thus, the method abovecan be employed with the target polynucleotide sequence being thesequence which spans the fusion. A method of treatment or prophylaxis ofsuch a cancer in a mammal comprises administering to the mammal acomposition of this invention wherein the target polynucleotide is apolynucleotide sequence of an abnormal cancer-causing gene in a mammalwhich also possesses a normal copy of the gene, and wherein thedifferences between the abnormal gene and the normal gene aredifferences in polynucleotide sequence. The skilled worker is familiarwith a large number of potential target genes for cancer therapy (forexample oncogenes such as ABL1, BCL1, BCL2, BCL6, CBFA2, CBL, CSF1R,ERBA, ERBB, EBRB2, FGR, FOS, FYN, HRAS, JUN, LCK, LYN, MYB, MYC, NRAS,RET or SRC; tumor suppressor genes such as BRCA1 or BRCA2; adhesionmolecules; cyclin kinases and their inhibitors). An exemplary list ofpotential target genes, including developmental genes, oncogenes, andenzymes, and a list of cancers that can be treated according to thepresent invention can be found in WO 99/32619. A candidate target genederived from a pathogen might, for example, cause immunosuppression ofthe host or be involved in replication of the pathogen, transmission ofthe pathogen, or maintenance of the infection.

Another embodiment of the invention provides a method for the treatmentof cancer (e.g., local and metastatic breast, ovarian, or porostatecancer) comprising: administration to the patient expression constructor vector (or a variant thereof) of the invention containing a cytotoxicgene.

Angiogenesis and suppressed immune response play a central role in thepathogenesis of malignant disease and tumor growth, invasion, andmetastasis. Thus, preferably, the therapeutic polypeptide has an abilityto induce an immune response and/or an anti-angiogenic response in vivo.In one embodiment, a gene therapy construct of the present inventionencodes a therapeutic gene that displays both immunostimulatory andanti-angiogenic activities, for example, IL12 (see Dias et al. (1998)Int J Cancer 75(1):151-157, and references cited herein below),interferon-alpha (O'Byrne et al. (2000) Eur J Cancer 36(2):151-169, andreferences cited therein), or a chemokine (Nomura & Hasegawa (2000)Anticancer Res 20(6A):4073-4080, and references cited therein). Inanother embodiment, a gene therapy construct of the present inventionencodes a gene product with immunostimulatory activity and a geneproduct having anti-angiogenic activity. See, e.g. Narvaiza et al.(2000) J Immunol 164:3112-3122. In another embodiment, the inventioncomprises a gene therapy construct encoding an IL2 polypeptide. IL12 isan immunostimulatory molecule that shows therapeutic activity in avariety of cancers, including renal cancer, breast cancer, bladdercancer, and malignant melanoma. The anti-tumor activity of IL2 isrelated to its capacity to expand and activate NK cells and T cells thatexpress IL2 receptors. See, e.g., Margolin (2000) Semin Oncol27(2):194-203; Gore (1996) Cancer Biother Radiopharm 11 (5):281-283;Deshmukh et al. (2001) J Neurosurgery 94(2):287-292; Larchian et al.(2000) Clin Cancer Res 6(7):2913-2920; Horiguchi et al. (2000) Gene Ther7(10):844-851; and references cited therein. IL2 has also been usedsuccessfully when co-administered with anti-tumor vaccines. See Overwijket al. (2000) Cancer J Sci Am 6 Suppl 1:S76-80, and references citedtherein.

3.2.2 Formulations and Administration

The chimeric RNA of the invention may be used and applied directly to ananimal or human in need of therapy or prophylaxis or may be appliedindirectly by means of an expression vector or construct.

3.2.2.1 Viral Gene Therapy Vectors

The present invention also provides gene therapy constructs or vectors.The particular vector employed in accordance with the methods of thepresent invention is not intended to be a limitation of the method forheat-induced expression of therapeutic genes by hyperthermia. Thus, anysuitable vector for delivery of the gene therapy construct can be used.

The vector can be a viral vector or a non-viral vector. Suitable viralvectors include adenoviruses, adeno-associated viruses (AAVs),retroviruses, pseudotyped retroviruses, herpes viruses, vacciniaviruses, Semiliki forest virus, and baculoviruses. Suitable non-viralvectors comprise plasmids, water-oil emulsions, polethylene imines,dendrimers, micelles, microcapsules, liposomes, and cationic lipids.Polymeric carriers for gene therapy constructs can be used as describedin Goldman et al (1997) Nat Biotechnol 15:462 and U.S. Pat. Nos.4,551,482 and 5,714,166. Peptide carriers are described in U.S. Pat. No.5,574,172. Where appropriate, two or more types of vectors can be usedtogether. For example, a plasmid vector can be used in conjunction withliposomes. Currently, a preferred embodiment of the present inventionenvisions the use of an adenovirus, a plasmid, or a liposome, eachdescribed further herein below.

As desired, vectors, especially viral vectors, can be selected toachieve integration of the nucleic acid of the construct of theinvention, into the genome of the cells to be transformed ortransfected. Including a ligand in the complex having affinity for aspecific cellular marker can also enhance delivery of the complexes to atarget in vivo. Ligands include antibodies, cell surface markers, viralpeptides, and the like, which act to home the complexes to tumorvasculature or endothelial cells associated with tumor vasculature, orto tumor cells themselves. A complex can comprise a construct or asecreted therapeutic polypeptide encoded by a construct. An antibodyligand can be an antibody or antibody fragment specific towards a tumormarker such as Her2/neu (v-erb-b2 avian erythroblastic leukemia viraloncogene homologue 2), CEA (carcinoembryonic antigen), ferritinreceptor, or a marker associated with tumor vasculature (integrins,tissue factor, or beta.-fibronectin isoform). Antibodies or otherligands can be coupled to carriers such as liposomes and viruses, as isknown in the art. See, e.g., Neri et al. (1997) Nat BioTechnology15:1271; Kirpotin et al. (1997) Biochemistry 36:66; Cheng (1996) HumanGene Therapy 7:275; Pasqualini et al. (1997) Nat Biotechnology 15:542;Park et al. (1997) Proc Am Ass Canc Res 38:342 (1997); Nabel (1997)“Vectors for Gene Therapy” in Current Protocols in Human Genetics onCD-ROM, John Wiley & Sons, New York, N.Y.; U.S. Pat. No. 6,071,890; andEuropean Patent No. 0 439 095. Alternatively, pseudotyping of aretrovirus can be used to target a virus towards a particular cell(Marin et al. (1997) Mol Med Today 3:396).

Viral vectors of the invention are preferably disabled, e.g.replication-deficient. That is, they lack one or more functional genesrequired for their replication, which prevents their uncontrolledreplication in vivo and avoids undesirable side effects of viralinfection. Preferably, all of the viral genome is removed except for theminimum genomic elements required to package the viral genomeincorporating the therapeutic gene into the viral coat or capsid. Forexample, it is desirable to delete all the viral genome except the LongTerminal Repeats (LTRs) or Invented Terminal Repeats (ITRs) and apackaging signal. In the case of adenoviruses, deletions are typicallymade in the E1 region and optionally in one or more of the E2, E3 and/orE4 regions. In the case of retroviruses, genes required for replication,such as env and/or gag/pol can be deleted. Deletion of sequences can beachieved by recombinant means, for example, involving digestion withappropriate restriction enzymes, followed by religation.Replication-competent self-limiting or selfdestructing viral vectors canalso be used.

Nucleic acid constructs of the invention can be incorporated into viralgenomes by any suitable means known in the art. Typically, suchincorporation will be performed by ligating the construct into anappropriate restriction site in the genome of the virus. Viral genomescan then be packaged into viral coats or capsids by any suitableprocedure. In particular, any suitable packaging cell line can be usedto generate viral vectors of the invention. These packaging linescomplement the replication-deficient viral genomes of the invention, asthey include, typically incorporated into their genomes, the genes whichhave been deleted from the replication-deficient genome. Thus, the useof packaging lines allows viral vectors of the invention to be generatedin culture.

Suitable packaging lines for retroviruses include derivatives of PA317cells, .psi.-2 cells, CRE cells, GRIP cells, E-86-GP cells, and 293GPcells. Line 293 cells can be used for adenoviruses and adeno-associatedviruses.

Suitable methods for introduction of a gene therapy construct into cellsinclude direct injection into a cell or cell mass, particle-mediatedgene transfer, electroporation, DEAEDextran transfection,liposome-mediated transfection, viral infection, and combinationsthereof. A delivery method is selected based considerations such as thevector type, the toxicity of the encoded gene, and the condition to betreated.

3.2.2.2 Suitable Expression Constructs

Various promoters can be employed to express the chimeric RNA moleculeof the invention to achieve a beneficial therapeutic or prophylacticeffect. By the methods and subject matter of the invention providedherein the expression becomes more specific, which is preferablyenhancing the beneficial effects and decreasing the side effects.

Various promoters are currently used in the art to express sequences inanimal, mammalian or human organism. Most of them are lackingtissue-specificity and can be advantageously combined with the teachingprovided herein. For example the promoter may be selected from groupconsisting of the perbB2 promoter, whey acidic protein promoter,stromelysin 3 promoter, prostate specific antigen promoter, probasinpromoter.

The promoter may be a heat or light inducible promoter, or chemicallyinducible promoter (e.g., a promoter inducible by antibiotic(tetracycline or its derivatives), acting on a fusion protein with atetracycline-responsive element).

The constructs may also comprise a heat-inducible promoter. Anyheat-inducible promoter can be used in accordance with the methods ofthe present invention, including but not limited to a heat-responsiveelement in a heat shock gene (e.g., hsp20-30, hsp27, hsp40, hsp60,hsp70, and hsp90). See Easton et al. (2000) Cell Stress Chaperones5(4):276-290; Csermely et al. (1998) Pharmacol Ther 79(2):129-168;Ohtsuka & Hata (2000) Int J Hyperthermia 16(3):231-245; and referencescited therein. Sequence similarity to heat shock proteins andheat-responsive promoter elements have also been recognized in genesinitially characterized with respect to other functions, and the DNAsequences that confer heat inducibility are suitable for use in thedisclosed gene therapy vectors. For example, expression ofglucose-responsive genes (e.g., grp94, grp78, mortalin/grp75) (Merricket al. (1997) Cancer Lett 119(2):185-190; Kiang et al. (1998) FASEB J12(14):1571-16-579), calreticulin (Szewczenko-Pawlikowski et al. (1997)Mol Cell Biochem 177(1-2):145-152); clusterin (Viard et al. (1999) JInvest Dermatol 112(3):290-296; Michel et al. (1997) Biochem J328(Ptl):45-50; Clark & Griswold (1997) J Androl 18(3):257-263),histocompatibility class I gene (HLA-G) (Ibrahim et al. (2000) CellStress Chaperones 5(3):207-218), and the Kunitz protease isoform ofamyloid precursor protein (Shepherd et al. (2000) Neuroscience99(2):317-325) are up-regulated in response to heat.

In the case of clusterin, a 14 base pair element that is sufficient forheat-inducibility has been delineated (Michel et al. (1997) Biochem J328(Pt1):45-50). Similarly, a twosequence unit comprising a 10- and a14-base pair element in the calreticulin promoter region has been shownto confer heat-inducibility (Szewczenko-Pawlikowski et al. (1997) MolCell Biochem 177(1-2):145-152).

Other promoter responsive to non-heat stimuli that can be used. Forexample, the mortalin promoter is induced by low doses of ionizingradiation (Sadekova (1997) Int J Radiat Biol 72(6):653-660), the hsp27promoter is activated by 17.beta.-estradiol and estrogen receptoragonists (Porter et al. (2001) J Mol Endocrinol 26(1):31-42), the HLA-Gpromoter is induced by arsenite, hsp promoters can be activated byphotodynamic therapy (Luna et al. (2000) Cancer Res 60(6):1637-1644).

A suitable promoter can incorporate factors such as tissue-specificactivation. For example, hsp70 is transcriptionally impaired in stressedneuroblastoma cells (Drujan & De Maio (1999) 12(6):443-448). Themortalin promoter, which is up-regulated in human brain tumors (Takanoet al. (1997) Exp Cell Res 237(1):38-45). A promoter employed in methodsof the present invention can show selective up-regulation in tumor cellsas described, for example, for mortalin (Takano et al. (1997) Exp CellRes 237(1):38-45), hsp27 and calreticulin (Szewczenko-Pawlikowski et al.(1997) Mol Cell Biochem 177(1-2):145-152; Yu et al. (2000)Electrophoresis 21(14):3058-3068), grp94 and grp78 (Gazit et al. (1999)Breast Cancer Res Treat 54(2):135-146), hsp27, hsp70, hsp73, and hsp90(Cardillo et al. (2000) Anticancer Res 20(6B):4579-4583; Strik et al.(2000) Anticancer Res 20(6B):4457-4552).

3.2.2.3 Formulations for Uptake of RNA and DNA

For the purpose of pharmaceutical applications it is preferred that thechimeric RNA molecule of the invention is applied or administered to thetarget cell or organism directly. Various means for application of RNAas pharmaceutical active ingredient are described in the art.

As used herein “administration” refers to contacting cells (e.g., eitherin isolated form or comprised in an organism) with the pharmaceuticalagent and can be performed in vitro or in vivo. With respect to in vivoapplications, the formulations of the present invention can beadministered to a patient in a variety of forms adapted to the chosenroute of administration, e.g., parenterally, orally, orintraperitoneally. Parenteral administration, which is preferred,includes administration by the following routes: intravenous;intramuscular; interstitially; intraarterially; subcutaneous; intraocular; intrasynovial; trans epithelial, including transdermal;pulmonary via inhalation; ophthalmic; sublingual and buccal; topically,including ophthalmic; dermal; ocular; rectal; and nasal inhalation viainsufflation. Preferably pharmaceutical preparations for the variousways of administration (such as parenteral, transmucosal, transdermal,oral, or topical application) are well known in the art and for exampledescribed in US Patent Application No. 20040014956. The pharmaceuticalagent of the invention may be administered systemically to a subject.Systemic absorption refers to the entry of drugs into the blood streamfollowed by distribution throughout the entire body. Administrationroutes which lead to systemic absorption include: intravenous,subcutaneous, intraperitoneal, and intranasal. The chosen method ofdelivery will result in entry into cells. Preferred delivery methodsinclude liposomes (10-400 nm), hydrogels, controlled-release polymers,and other pharmaceutically applicable vehicles, and microinjection orelectroporation (for ex vivo treatments). Drug delivery vehicles can bechosen e.g., for in vitro, for systemic, or for topical administration.These vehicles can be designed to serve as a slow release reservoir orto deliver their contents directly to the target cell. An advantage ofusing some direct delivery drug vehicles is that multiple molecules aredelivered per uptake. Some examples of such specialized drug deliveryvehicles which fall into this category are liposomes, hydrogels,cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres.In one embodiment, in vitro treatment of cells with oligonucleotides canbe used for ex vivo therapy of cells removed from a subject (e.g., fortreatment of leukemia or viral infection) or for treatment of cellswhich did not originate in the subject, but are to be administered tothe subject (e.g., to eliminate transplantation antigen expression oncells to be transplanted into a subject). In addition, in vitrotreatment of cells can be used in non-therapeutic settings, e.g., toevaluate gene function, to study gene regulation and protein synthesisor to evaluate improvements made to oligonucleotides designed tomodulate gene expression or protein synthesis. In vivo treatment ofcells can be useful in certain clinical settings where it is desirableto inhibit the expression of a protein.

Compositions for pharmaceutical use of this invention desirably containa chimeric RNA molecule, or an expression construct for its production(hereinafter the “pharmaceutical agent”). Any of the pharmaceuticalagents can be used alone or in conjunction with a pharmaceuticallyacceptable carrier and with additional optional components forpharmaceutical delivery. As used herein, “pharmaceutically acceptablecarrier” includes appropriate solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like. The use of such media and agents forpharmaceutical active substances is well known in the art. Suitablepharmaceutically acceptable carriers facilitate administration of thepolynucleotide compositions of this invention, but are physiologicallyinert and/or nonharmful. Carriers may be selected by one of skill in theart. Such carriers include but are not limited to, sterile saline,phosphate, buffered saline, dextrose, sterilized water, glycerol,ethanol, lactose, sucrose, calcium phosphate, gelatin, dextran, agar,pectin, peanut oil, olive oil, sesame oil, and water and combinationsthereof Additionally, the carrier or diluent may include a time delaymaterial, such as glycerol monostearate or glycerol distearate alone orwith a wax. In addition, slow release polymer formulations can be used.The formulation should suit not only the form of the delivery agent, butalso the mode of administration. Selection of an appropriate carrier inaccordance with the mode of administration is routinely performed bythose skilled in the art. Additional components for the carrier mayinclude but are not limited to adjuvants, preservatives, chemicalstabilizers, or other antigenic proteins. Suitable exemplarypreservatives include chlorobutanol, potassium sorbate, sorbic acid,sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin,phenol, and parachlorophenol. Suitable stabilizing ingredients which maybe used include, for example, casamino acids, sucrose, gelatin, phenolred, N-Z amine, monopotassium diphosphate, lactose, lactalbuminhydrolysate, and dried milk. A conventional adjuvant is used to attractleukocytes or enhance an immune response. Such adjuvants include, amongothers, Ribi, mineral oil and water, aluminum hydroxide, Amphigen,Avridine, L121/squalene, D-lactide-polylactide/glycoside, pluronicplyois, muramyl dipeptide, killed Bordetella, and saponins, such as QuilA.

The pharmaceutical agent may be incorporated into liposomes or liposomesmodified with polyethylene glycol or admixed with cationic lipids forparenteral administration. Incorporation of additional substances intothe liposome, for example, antibodies reactive against membrane proteinsfound on specific target cells, can help target the oligonucleotides tospecific cell types. Liposomes can be prepared by any of a variety oftechniques that are known in the art. See e.g., Betageri et al. (1993)Liposome Drug Delivery Systems, Technomic Publishing, Lancaster;Gregoriadis, ed. (1993) Liposome Technology, CRC Press, Boca Raton,Fla.; Janoff, ed. (1999) Liposomes: Rational Design, M. Dekker, NewYork, N.Y.; Lasic & Martin (1995) Stealth Liposomes, CRC Press, BocaRaton, Fla.; Nabel (1997) “Vectors for Gene Therapy” in CurrentProtocols in Human Genetics on CD-ROM, John Wiley & Sons, New York,N.Y.; and U.S. Pat. Nos. 4,235,871; 4,551,482; 6,197,333; and 6,132,766.Entrapment of an active agent within liposomes of the present inventioncan also be carried out using any conventional method in the art. Inpreparing liposome compositions, stabilizers such as antioxidants andother additives can be used. Other lipid carriers can also be used inaccordance with the claimed invention, such as lipid microparticles,micelles, lipid suspensions, and lipid emulsions. See, e.g.,Labat-Moleur et al. (1996) Gene Therapy 3:1010-1017; U.S. Pat. Nos.5,011,634; 6,056,938; 6,217886; 5,948,767; and 6,210,707.

The composition of the invention may also involve lyophilizedpolynucleotides, which can be used with other pharmaceuticallyacceptable excipients for developing powder, liquid or suspension dosageforms, including those for intranasal or pulmonary applications. See,e.g., Remington: The Science and Practice of Pharmacy, Vol. 2, 19.sup.thedition (1995), e.g., Chapter 95 Aerosols; and International PatentApplication No. PCT/US99/05547, the teachings of which are herebyincorporated by reference.

In some preferred embodiments, the pharmaceutical compositions of theinvention are prepared for administration to mammalian subjects in theform of, for example, liquids, emulsions, powders, aerosols, tablets,capsules, enteric coated tablets or capsules, or suppositories. Theoptimal course of administration or delivery of the pharmaceutical agentmay vary depending upon the desired result and/or on the subject to betreated.

The pharmaceutical preparations of the present invention may be preparedand formulated as emulsions or microemulsions. Emulsions are usuallyheterogenous systems of one liquid dispersed in another in the form ofdroplets usually exceeding 0.1 μm in diameter. The emulsions of thepresent invention may contain excipients such as emulsifiers,stabilizers, dyes, fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives, andanti-oxidants may also be present in emulsions as needed. Theseexcipients may be present as a solution in either the aqueous phase,oily phase or itself as a separate phase. Suitable examples foremulsifiers and preservatives are given in US Patent Application No.20040014956. A microemulsion is a system of water, oil and amphiphilewhich is a single optically isotropic and thermodynamically stableliquid solution. Typically microemulsions are prepared by firstdispersing an oil in an aqueous surfactant solution and then adding asufficient amount of a 4th component, generally an intermediatechain-length alcohol to form a transparent system. Suitable examples forsurfanctants and cosurfactants are described in US Patent ApplicationNo. 20040014956. Microemulsions are particularly of interest from thestandpoint of drug solubilization and the enhanced absorption of drugs.Lipid based microemulsions (both oil/water and water/oil) have beenproposed to enhance the oral bioavailability of drugs. It is expectedthat the microemulsion compositions and formulations of the presentinvention will facilitate the increased systemic absorption ofpharmaceutical agents of the invention from the gastrointestinal tract,as well as improve the local cellular uptake of oligonucleotides withinthe gastrointestinal tract, vagina, buccal cavity and other areas ofadministration.

In an embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of the pharmaceutical agentsof the invention (especially nucleic acids, particularlyoligonucleotides) to the skin of humans and animals. Suitablepenetration enhancer are described in US Patent Application NO.20040146902, herein incorporated by reference.

The useful dosage to be administered and the particular mode ofadministration will vary depending upon such factors as the cell type,or for in vivo use, the age, weight and the particular animal and regionthereof to be treated, the particular oligonucleotide and deliverymethod used, the therapeutic or diagnostic use contemplated, and theform of the formulation, for example, suspension, emulsion, micelle orliposome, as will be readily apparent to those skilled in the art.Typically, dosage is administered at lower levels and increased untilthe desired effect is achieved. The dosage of the pharmaceutical agentmay be adjusted to optimally reduce expression from the target gene,e.g., as measured by a readout of RNA stability or by a therapeuticresponse, without undue experimentation. The exact dosage of anoligonucleotide and number of doses administered will depend upon thedata generated experimentally and in clinical trials. Several factorssuch as the desired effect, the delivery vehicle, disease indication,and the route of administration, will affect the dosage. Dosages can bereadily determined by one of ordinary skill in the art and formulatedinto the subject pharmaceutical compositions. Preferably, the durationof treatment will extend at least through the course of the diseasesymptoms. For example, the compositions of the present invention, whenused as pharmaceutical compositions, can comprise about 1 ng to about 20mgs of the pharmaceutical agent of the invention (e.g., the syntheticRNA molecules or the delivery agents which can be DNA molecules,plasmids, viral vectors, recombinant viruses, and mixtures thereof). Thecompositions of the present invention in which the delivery agents aredonor cells or bacterium can be delivered in dosages of between about 1cell to about 10⁷ cells/dose. Similarly, where the delivery agent is alive recombinant virus, a suitable vector-based composition containsbetween 1×10² pfu to 1×10¹² pfu per dose.

The pharmaceutical agent of the invention may be combined with any otherdrug, preferably for the same medicinal indication. For example forpharmaceutical agents which have anti-cancer properties the agent may becombined with one or more chemotherapeutic agents (e.g., such asdaunorubicin, idarubicin, mitomycin C, 5-fluorouracil (5-FU),methotrexate (MTX), taxol, vincristine, and cisplatin) that function bya non-antisense mechanism.

Additional suitable teachings for pharmaceutical compositions and theirpreparation, administration and dosing in relation to oligonucleotidecompounds which may be utilized within the scope of the presentinvention are given in US Patent Application No. 20040146902

In one embodiment, the pharmaceutical agents of the invention (e.g.,oligonucleotides) can be administered to subjects. Examples of subjectsinclude mammals, e.g., humans and other primates; cows, pigs, horses,and farming (agricultural) animals; dogs, cats, and other domesticatedpets; mice, rats, and transgenic non-human animals.

3.3. Biotechnological Applications

The methods and compositions according to the invention can be appliedadvantageously in biotechnological applications and methods, includingbut not limited to optimization of metabolic pathways e.g., in yeasts,fungi or other eukaryotic microorganisms or cells which are used infermentation for the production of fine chemicals such as amino acids(for example lysin or methionin), vitamins (such as vitamin B2, vitaminC, vitamin E), carotenoids, oils and fats, polyunsaturated fatty acids,biotin and the like.

Preferred vectors for expression in eukaryotes comprise pWLNEO, pSV2CAT,pOG44, pXT1 and pSG (Stratagene Inc.); pSVK3, pBPV, pMSG and pSVL(Pharmacia Biotech, Inc.). Inducible vectors which may be mentioned arepTet-tTak, pTet-Splice, pcDNA4/TO, pcDNA4/TO/LacZ, pcDNA6/TR,pcDNA4/TO/Myc-His/LacZ, pcDNA4/TO/Myc-His A, pcDNA4/TO/Myc-His B,pcDNA4/TO/Myc-His C, pVgRXR (Invitrogen, Inc.) or the pMAM series(Clontech, Inc.; GenBank Accession No.: U02443). These vectors alreadyprovide the inducible regulatory control element, for example for achemically inducible expression of a DSBI enzyme. The nucleic acidsequence encoding a DSBI enzyme can be inserted directly into thesevectors. Vectors for expression in yeast comprise for example pYES2,pYD1, pTEFI/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalph, pPIC9, pPIC3.5,PHIL-D2, PHIL-SI, pPIC3SK, pPIC9K and PA0815 (Invitrogen, Inc.). Inprinciple, for the transformation of animal cell or of yeast cells,similar methods as the “direct” transformation of plant cells are to beapplied. In particular, methods such as the calcium-phosphate- orliposome-mediated transformation or else electroporation are preferred.Selection markers which can be used are, in principle, many of theselection systems which are also preferred for plants. Especiallypreferred are for mammalian cell the neomycin (G418) resistance, thehygromycin resistance, the zeocin resistance or the puromycinresistance. The ampicillin resistance, the kanamycin resistance or thetetracycline resistant are especially preferred for prokaryotes.

Depending on the host organism, the organisms used in the method aregrown or cultured in a manner with which the skilled worker is familiar.As a rule, microorganisms are grown in a liquid medium comprising acarbon source, usually in the form of sugars, a nitrogen source, usuallyin the form of organic nitrogen sources such as yeast extracts or saltssuch as ammonium sulfate, trace elements such as salts of iron,manganese and magnesium, and, if appropriate, vitamins, at temperaturesof between 0° C. and 100° C., preferably between 10° C. to 60° C., whilepassing in oxygen. The pH of the liquid medium can be kept at a constantvalue, that is to say regulated during the culturing period, or elsenot. The culture can be batchwise, semibatchwise or continuous.Nutrients can be provided at the beginning of the fermentation or fed insemicontinuously or continuously.

4. Exemplification

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims. The entire contentsof all patents, published patent applications and other references citedherein are hereby expressly incorporated herein in their entireties byreference.

The invention, now being generally described, will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention and are not intended to limit the invention.

EXAMPLES General Methods

Unless otherwise specified, all chemicals are obtained from Fluka(Buchs), Merck (Darmstadt), Roth (Karlsruhe), Serva (Heidelberg) andSigma (Deisenhofen). Restriction enzymes, DNA-modifying enzymes andmolecular biology kits were from AmershamPharmacia (Freiburg), Biometra(Göttingen), Roche (Mannheim), New England Biolabs (Schwalbach), Novagen(Madison, Wis., USA), Perkin-Elmer (Weiterstadt), Qiagen (Hilden),Stratagen (Amsterdam, Netherlands), Invitrogen (Karlsruhe) and Ambion(Cambridgeshire, United Kingdom). The reagents used were employed inaccordance with the manufacturer's instructions.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, microbiology, recombinant DNA, and immunology, whichare within the skill of the art. Such techniques are explained fully inthe literature. See, for example, Molecular Cloning A Laboratory Manual,2nd Ed., ed. by Sambrook, J. et al. (Cold Spring Harbor Laboratory Press(1989)); Short Protocols in Molecular Biology, 3rd Ed., ed. by Ausubel,F. et al. (Wiley, N.Y. (1995)); DNA Cloning, Volumes I and II (D. N.Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed. (1984));Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D.Hames & S. J. Higgins eds. (1984)); the treatise, Methods In Enzymology(Academic Press, Inc., N.Y.); Immunochemical Methods In Cell AndMolecular Biology (Mayer and Walker, eds., Academic Press, London(1987)); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weirand C. C. Blackwell, eds. (1986)); and Miller, J. Experiments inMolecular Genetics (Cold Spring Harbor Press, Cold Spring Harbor, N.Y.(1972)).

The chemical synthesis of oligonucleotides can be carried out forexample in the known manner using the phosphoamidite method (Voet, Voet,2nd edition, Wiley Press New York, pages 896-897). The cloning stepscarried out for the purpose of the present invention such as, forexample, restriction cleavages, agarose gel electrophoresis,purification of DNA fragments, transfer of nucleic acids tonitrocellulose and nylon membranes, linking DNA fragments,transformation of E. coli cells, bacterial cultures, propagation ofphages and sequence analysis of recombinant DNA, are carried out asdescribed in Sambrook et al. (1989) Cold Spring Harbor Laboratory Press;ISBN 0-87969-309-6. Recombinant DNA molecules are sequenced using an ABIlaser fluorescence DNA sequencer by the method of Sanger (Sanger et al.(1977) Proc Natl Acad Sci USA 74:5463-5467).

Example 1: Agrobacterium-Mediated Transformation in Dicotyledonous andMonocotyledonous Plants

1.1 Transformation and Regeneration of Transgenic Arabidopsis thaliana(Columbia) Plants

To generate transgenic Arabidopsis plants, Agrobacterium tumefaciens(strain C58C1 pGV2260) is transformed with various ptxA or SbHRGP3promoter/GUS vector constructs. The agrobacterial strains aresubsequently used to generate transgenic plants. To this end, a singletransformed Agrobacterium colony is incubated overnight at 28° C. in a 4mL culture (medium: YEB medium with 50 μg/mL kanamycin and 25 μg/mLrifampicin). This culture is subsequently used to inoculate a 400 mLculture in the same medium, and this is incubated overnight (28° C., 220rpm) and spun down (GSA rotor, 8,000 rpm, 20 min). The pellet isresuspended in infiltration medium (1/2 MS medium; 0.5 g/L MES, pH 5.8;50 g/L sucrose). The suspension is introduced into a plant box(Duchefa), and 100 mL of SILWET L-77 (heptamethyltrisiloxan modifiedwith polyalkylene oxide; Osi Specialties Inc., Cat. P030196) was addedto a final concentration of 0.02%. In a desiccator, the plant box with 8to 12 plants is exposed to a vacuum for 10 to 15 minutes, followed byspontaneous aeration. This is repeated twice or 3 times. Thereupon, allplants are planted into flowerpots with moist soil and grown underlong-day conditions (daytime temperature 22 to 24° C., nighttimetemperature 19° C.; relative atmospheric humidity 65%). The seeds areharvested after 6 weeks.

As an alternative, transgenic Arabidopsis plants can be obtained by roottransformation. White root shoots of plants with a maximum age of 8weeks are used. To this end, plants which are kept under sterileconditions in 1 MS medium (1% sucrose; 100 mg/L inositol; 1.0 mg/Lthiamine; 0.5 mg/L pyridoxine; 0.5 mg/L nicotinic acid; 0.5 g MES, pH5.7; 0.8% agar) are used. Roots are grown on callus-inducing medium for3 days (1× Gamborg's B5 medium; 2% glucose; 0.5 g/L mercaptoethanol;0.8% agar; 0.5 mg/L 2,4-D (2,4-dichlorophenoxyacetic acid); 0.05 mg/Lkinetin). Root sections 0.5 cm in length are transferred into 10 to 20mL of liquid callus-inducing medium (composition as described above, butwithout agar supplementation), inoculated with 1 mL of theabove-described overnight agrobacterial culture (grown at 28° C., 200rpm in LB) and shaken for 2 minutes. After excess medium has beenallowed to run off, the root explants are transferred to callus-inducingmedium with agar, subsequently to callus-inducing liquid medium withoutagar (with 500 mg/L betabactyl, SmithKline Beecham Pharma GmbH, Munich),incubated with shaking and finally transferred to shoot-inducing medium(5 mg/L 2-isopentenyladenine phosphate; 0.15 mg/L indole-3-acetic acid;50 mg/L kanamycin; 500 mg/L betabactyl). After 5 weeks, and after 1 or 2medium changes, the small green shoots are transferred to germinationmedium (1 MS medium; 1% sucrose; 100 mg/L inositol; 1.0 mg/L thiamine;0.5 mg/L pyridoxine; 0.5 mg/L nicotinic acid; 0.5 g MES, pH 5.7; 0.8%agar) and regenerated into plants.

1.2 Transformation and Regeneration of Crop Plants

The Agrobacterium-mediated plant transformation using standardtransformation and regeneration techniques may also be carried out forthe purposes of transforming crop plants (Gelvin & Schilperoort (1995)Plant Molecular Biology Manual, 2nd Edition, Dordrecht: Kluwer, AcademicPubl. ISBN 0-7923-2731-4; Glick & Thompson (1993) Methods in PlantMolecular Biology and Biotechnology, Boca Raton: CRC Press, ISBN0-8493-5164-2)

For example, oilseed rape can be transformed by cotyledon or hypocotyltransformation (Moloney et al. (1989) Plant Cell Reports 8: 238-242, deBlock et al. (1989) Plant Physiol. 91:694-701) The use of antibioticsfor the selection of Agrobacteria and plants depends on the binaryvector and the Agrobacterium strain used for the transformation. Theselection of oilseed rape is generally carried out using kanamycin asselectable plant marker. The Agrobacterium-mediated gene transfer inlinseed (Linum usitatissimum) can be carried out using for example atechnique described by Mlynarova et al. ((1994), Plant Cell Report 13:282-285). The transformation of soya can be carried out using, forexample, a technique described in EP-A1 0424 047 or in EP-A1 0397 687,U.S. Pat. Nos. 5,376,543, 5,169,770. The transformation of maize orother monocotyledonous plants can be carried out using, for example, atechnique described in U.S. Pat. No. 5,591,616.

The transformation of plants using particle bombardment, polyethyleneglycol-mediated DNA uptake or via the silicon carbonate fiber techniqueis described, for example, by Freeling & Walbot (1993) “The maizehandbook” ISBN 3-540-97826-7, Springer Verlag New York).

Example 2: Detection of Reporter Gene Expression

These experiments are performed by bombardment of plant tissues orculture cells (Example 2.1), by PEG-mediated (or similar methodology)introduction of DNA to plant protoplasts (Example 2.2), or byAgrobacterium-mediated transformation (Example 2.3). The target tissuefor these experiments can be plant tissues (e.g. leaf tissue has beendescribed to best support IRES-mediated translation (Urwin, et al.,2000), cultured plant cells (e.g. maize BMS), or plant embryos forAgrobacterium protocols.

2.1 Transient Assay Using Microprojectile Bombardment

The plasmid constructs are isolated using Qiagen plasmid kit (cat#12143). DNA is precipitated onto 0.6 μM gold particles (Bio-Rad cat#165-2262) according to the protocol described by Sanford et al. (1993)and accelerated onto target tissues (e.g. two week old maize leaves, BMScultured cells, etc.) using a PDS-1000/He system device (Bio-Rad). AllDNA precipitation and bombardment steps are performed under sterileconditions at room temperature.

Two mg of gold particles (2 mg/3 shots) are resuspended in 100% ethanolfollowed by centrifugation in a Beckman Microfuge 18 Centrifuge at 2000rpm in an Eppendorf tube. The pellet is rinsed once in sterile distilledwater, centrifuged, and resuspended in 25 μL of 1 μg/μL total DNA. Thefollowing reagents are added to the tube: 220 μL H₂0, 250 μL 2.5M CaCl₂,50 μL 0.1M spermidine, freebase. The DNA solution is briefly vortexedand placed on ice for 5 min followed by centrifugation at 500 rpm for 5min in a Beckman Microfuge 18 Centrifuge. The supernatant is removed.The pellet is resuspended in 600 μL ethanol followed by centrifugationfor 1 min at 14,000 rpm. The final pellet is resuspended in 36 μL ofethanol and used immediately or stored on ice for up to 4 hr prior tobombardment. For bombardment, two-week-old maize leaves are cut inapproximately 1 cm in length and located on 2 inches diamentersterilized Whatman filter paper. In the case of BMS cultured cells, 5 mLof one-week-old suspension cells are slowly vacuum filtered onto the 2inches diameter filter paper placed on a filter unit to remove excessliquid. The filter papers holding the plant materials are placed onosmotic induction media (N6 1-100-25, 0.2 M mannitol, 0.2 M sorbitol) at27° C. in darkness for 2-3 hours prior to bombardment. A few minutesprior to shooting, filters are removed from the medium and placed ontosterile opened Petri dishes to allow the calli surface to partially dry.To keep the position of plant materials, a sterilized wire mesh screenis laid on top of the sample. Each plate is shot with 10 μL of gold-DNAsolution once at 2,200 psi for the leaf materials and twice at 1100 psifor the BMS cultured cells. Following bombardment, the filters holdingthe samples are transferred onto MS basal media and incubated for 2 daysin darkness at 27° C. prior to transient assays. Transient expressionlevels of the reporter gene are determined quantification of expressionof reporter genes or RT-PCR using the protocols in the art in order todetermine potentially strong and tight terminator candidates.

2.2 Transient Assay Using Protoplasts

Isolation of protoplasts is conducted by following the protocoldeveloped by Sheen (1990). Maize seedlings are kept in the dark at 25°C. for 10 days and illuminated for 20 hours before protoplastpreparation. The middle part of the leaves are cut to 0.5 mm strips(about 6 cm in length) and incubated in an enzyme solution containing 1%(w/v) cellulose RS, 0.1% (w/v) macerozyme R10 (both from Yakult Honsha,Nishinomiya, Japan), 0.6 M mannitol, 10 mM Mes (pH 5.7), 1 mM CaCl₂, 1mM MgCl₂, 10 mM β-mercaptoethanol, and 0.1% BSA (w/v) for 3 hr at 23° C.followed by gentle shaking at 80 rpm for 10 min to release protoplasts.Protoplasts are collected by centrifugation at 100×g for 2 min, washedonce in cold 0.6 M mannitol solution, centrifuged, and resuspended incold 0.6 M mannitol (2×10⁶/mL). A total of 50 μg plasmid DNA in a totalvolume of 100 μL sterile water is added into 0.5 mL of a suspension ofmaize protoplasts (1×10⁶ cells/mL) and mix gently. 0.5 mL PEG solution(40% PEG 4000, 100 mM CaNO₃, 0.5 mannitol) is added and prewarmed at 70°C. with gentle shaking followed by addition of 4.5 mL MM solution (0.6 Mmannitol, 15 mM MgCl₂, and 0.1% MES). This mixture is incubated for 15minutes at room temperature. The protoplasts are washed twice bypelleting at 600 rpm for 5 min and resuspending in 1.0 mL of MMBsolution[0.6 M mannitol, 4 mM Mes (pH 5.7), and brome mosaic virus (BMV)salts (optional)] and incubated in the dark at 25° C. for 48 hr. Afterthe final wash step, collect the protoplasts in 3 mL MMB medium, andincubate in the dark at 25° C. for 48 hr. Transient expression levels ofthe reporter gene are determined quantification of expression ofreporter genes or RT-PCR using the protocols in the art in order todetermine potentially strong and tight terminator candidates.

2.3 Detection of GUS Reporter Gene

To identify the characteristics of the promoter and the essentialelements of the latter, which bring about its tissue specificity, it isnecessary to place the promoter itself and various fragments thereofbefore what is known as a reporter gene, which allows the determinationof the expression activity. An example, which may be mentioned, is thebacterial β-glucuronidase (Jefferson et al. EMBO J 6:3901-3907 (1987).The β-glucuronidase activity can be detected in-planta by means of achromogenic substrate such as 5-bromo-4-chloro-3-indolyl-β-D-glucuronicacid in an activity staining (Jefferson et al. Plant Mol Biol Rep5:387-405 (1987)). To study the tissue specificity, the plant tissue iscut, embedded, stained and analyzed as described (for example Bäumleinet al. (1991a) Mol Gen Genet 225(3):459-467, Bäumlein et al. (1991b) MolGen Genet 225:121-128).

A second assay permits the quantitative determination of the GUSactivity in the tissue studied. For the quantitative activitydetermination, MUG (4-methylumbelliferyl-β-D-glucuronide) is used assubstrate for β-glucuronidase, and the MUG is cleaved into MU(methylumbelliferone) and glucuronic acid.

To do this, a protein extract of the desired tissue is first preparedand the substrate of GUS is then added to the extract. The substrate canbe measured fluorimetrically only after the GUS has been reacted.Samples that are subsequently measured in a fluorimeter are taken atvarious points in time. This assay may be carried out for example withlinseed embryos at various developmental stages (21, 24 or 30 days afterflowering). To this end, in each case one embryo is ground into a powderin a 2 mL reaction vessel in liquid nitrogen with the aid of avibration-grinding mill (Type: Retsch MM 2000). After addition of 100 μLof EGL buffer (0.1 M KPO₄, pH 7.8; 1 mM EDTA; 5% glycerol; 1 M DTT), themixture is centrifuged for 10 minutes at 25° C. and 14,000×g. Thesupernatant is removed and recentrifuged. Again, the supernatant istransferred to a new reaction vessel and kept on ice until further use.25 μL of this protein extract are treated with 65 μL of EGL buffer(without DTT) and employed in the GUS assay. 10 μL of the substrate MUG(10 mM 4-methylumbelliferyl-β-D-glucuronide) are now added, the mixtureis vortexed, and 30 μL are removed immediately as zero value and treatedwith 470 μL of Stop buffer (0.2 M Na₂CO₃). This procedure is repeatedfor all of the samples at an interval of 30 seconds. The samples takenwere stored in the refrigerator until measured. Further readings weretaken after 1 h and after 2 h. A calibration series which containedconcentrations from 0.1 mM to 10 mM MU (4-methylumbelliferone) wasestablished for the fluorimetric measurement. If the sample values wereoutside these concentrations, less protein extract was employed (10 μL,1 μL, 1 μL from a 1:10 dilution), and shorter intervals were measured (0h, 30 min, 1 h). The measurement was carried out at an excitation of 365nm and an emission of 445 nm in a Fluoroscan II apparatus (Labsystem).As an alternative, the substrate cleavage can be monitoredfluorimetrically under alkaline conditions (excitation at 365 nm,measurement of the emission at 455 nm; Spectro Fluorimeter BMGPolarstar+) as described in Bustos et al. (1989) Plant Cell 1(9):839-53.All the samples were subjected to a protein concentration determinationby the method of Bradford (1976) Anal. Biochem. 72:248-254, thusallowing an identification of the promoter activity and promoterstrength in various tissues and plants.

2.4 Detection of Fluorescent Protein Gene

Several fluorescent protein genes, e.g. DsRed, ZsGreen, ZsYellow, ZsCyanand AcGFP (BD Biosciences) are derived from new species of reef coraland jelly fish. It has been shown that these fluorescent proteins can beused as reporters in multiple plant species (Wenck A. et al., Plant CellReport, 22:244-251, 2003). The plant materials (e.g. leaves and roots)carrying fluorescent proteins can be visualized using epifluoresecncemicroscope with appropriate filter sets. Furthermore, the intensity offluorescent protein, which indicates the expression level of theprotein, is analyzed by a fluorescence imaging instrument such asTyphoon 9400 (Amersham Biosciences) in a quantitative manner followingthe instruction recommended by the manufacturer.

Example 3: Expression Analysis for microRNAs

Analysis is performed on RNA-level (e.g., by Northern blot analysis orreal time qPCR). Alternatively expression profiles can be evaluated bythe representation of specific miRNA sequences in non-normalizedtissue-specific cDNA libraries and can—for example—be assessed in silicoby “counting” the number of cDNA sequences for a specific miRNA in saidlibrary.

3.1 Northern Hybridization:

A suitable method for determining the amount of transcription of a geneis to carry out a Northern blot analysis (by way of reference, seeAusubel et al. (1988) Current Protocols in Molecular Biology, Wiley: NewYork, or the abovementioned example section), where a primer which isdesigned in such a way that it binds to the gene of interest is labeledwith a detectable label (usually a radioactive label or chemiluminescentlabel) so that, when the total RNA of a culture of the organism isextracted, separated on a gel, transferred to a stable matrix andincubated with this probe, the binding and the extent of the binding ofthe probe indicate the presence and also the amount of the mRNA for thisgene. This information also indicates the degree of transcription of thetransformed gene. Cellular total RNA can be prepared from cells, tissuesor organs in a plurality of methods, all of which are known in the art,such as, for example, the method described by Bormann, E. R., et al.(1992) Mol. Microbiol. 6:317-326.

To carry out the RNA hybridization, 20 μg of total RNA or 1 μg ofpoly(A)⁺ RNA are separated by means of gel electrophoresis in agarosegels with a strength of 1.25% using formaldehyde, as described inAmasino (1986, Anal. Biochem. 152, 304), capillary-blotted to positivelycharged nylon membranes (Hybond N+, Amersham, Braunschweig) using10×SSC, immobilized by means of UV light and prehybridized for 3 hoursat 68° C. using hybridization buffer (10% dextran sulfate w/v, 1 M NaCl,1% SDS, 100 mg herring sperm DNA). The DNA probe was labeled with theHighprime DNA labeling kit (Roche, Mannheim, Germany) during theprehybridization, using alpha-³²P-dCTP (Amersham Pharmacia,Braunschweig, Germany). After the labeled DNA probe had been added, thehybridization was carried out in the same buffer at 68° C. overnight.The washing steps were carried out twice for 15 minutes using 2×SSC andtwice for 30 minutes using 1×SSC, 1% SDS, at 68° C. The sealed filterswere exposed at −70° C. over a period of 1 to 14 days.

3.2 RT-qPCR

After total RNA is isolated from an organism or specific tissues or celltypes, RNA is resolved on a denaturing 15% polyacrylamide gel. A gelfragment represents the size range of 15 to 26 nucleotides was excised,small RNA was eluted, and recovered. Subsequently, small RNA is ligatedto 5′ and 3′ RNA/DNA chimeric oligonucleotide adapters. Reversetranscription reaction was performed using RT primer followed by PCRwith appropriate primers. PCR products are then cloned into vector forsequencing (Sunkar R and Zhu J K, The Plant Cell 16:2001:2019, 2004)

3.3 Results

The following tables present some of the expression profiles found forvarious miRNAs both in plant and animal or mammalian species. Duringcloning and subsequent sequencing of miRNA, some miRNA-clones have showndifferent nucleotides at the ends (especially 3′-end), which arerepresented herein by small letters. The 3′ end of miRNA is usually lessimportant.

TABLE 2 miRNAs identified from Arabidopsis thaliana libraries.At pri-mlRNA ID At miR319b At miR160b At miR163 At miR167a At miR172bAt miRNA sequence UUG- UGCCUGCUCC UUGAAGAGGAC- UGAA- AGAAUCUUGAUGACUGAAGG CUGUAUGCCA UUGGAACU- GCUGCCAG- GAUGCUGCAU GAGCUCCC UCGAUCAUGAUCUA SEQ ID NO: 5 1 2 3 4 Hyseq clone ID 65631003 65987305 6561328864879045 Contig1562 Library Relative Relative Relative Relative RelativeLibrary Name Synonym Description Expression Expression ExpressionExpression Expression AC103 seedfill Developing siliques with 0 0 00.667 0 seeds 1 to 14 d post anthesis AC104 shootNormal rosettes prior to 0 0 0 0 0 bolting AC108 shootRosettes inoculated with 0 0.059 0 0 0 conidia of Erysiphecichoracearum, Blumeria f.sp. Hordei, Alternaria alternata,or A. brassicicloa for 12, 24, 48, 73 H AC109 flowerNormal flower bud and seed 0.333 0.235 0.714 0.333 0.778 developmentAC114 stress Mixed treatment: 1. 2 H dessication, 0 0.176 0.286 0 02. up to 6 H 300 mM NaCl, 3. Cold at −2 C., or 0 C.or 6 C., 4. 20 mM hydrogen peroxide. (1, 2, 3) had sometreatments allowing recovery. (1, 2) entire plants harvested,(3, 4) only shoots harvested. AC115 callus Callus (Initiated from seeds)0.667 0.176 0 0 0 minimally induced to form either roots (5 mg/L NAA +0.1 iP) or shoots (1 mg/L NAA + 0.1iP) AC117 root.mixRoots from aerated hydro- 0 0.353 0 0 0.111 ponics (continuous) withvarying nutrient strength. AC119 RNA Mix Mixed mRNA from all Arabidopsis0 0 0 0 0.111 libraris. Preferred Use Prevent leakinessPrevent leakiness Prevent leakiness Prevent leakiness Prevent in leafin root and in flowers in seeds and leakiness tissue flowers flowersin flowers

Table 3-A: miRNAs identified from Oryza sativa libraries. Os pri- miRNAOs Os Os Os Os Os Os Os Os Os ID miR167g miR168a miR169g miR169i miR171bmiR397b miR398a miR399k miR156l miR159b Os UGAAGCU UCGCUUG UAGCCAAUAGCCAA UGAUUGA UUAUUGA UGUGUUC UGCCAAA CGACAGA UUUGGAU miRNA GCCAGCAGUGCAGA GGAUGAC GGAUGAC GCCGUG GUGCAGC UCAGGUC GGAAAUU AGAGAGU UGAAGGGsequence UGAUCUg UCGGGAC UUgccua UUgccug CCAAUAUC GUUGAUG ACCCCUUUGCCCCG GAGCAUA AGCUCUG SEQ ID 14 15 16 17 18 19 20 21 7 8 NO: HyseqContig6503 Contig2277 Contig17418 3282464 37697372 Contig16437 37947875Contig10310 35003089 Contig4124 clone ID Library Library RelativeRelative Relative Relative Relative Relative Relative Relative RelativeRelative Name Synonym Description Expression Expression ExpressionExpression Expression Expression Expression Expression ExpressionExpression AC003 shoot Shoots 0.033 0.056 0.176 0.333 0 0.094 0 0.014 00.022 AC004 shoot Shoot meristems 0 0.062 0.235 0 0 0.019 0 0.007 0.50.267 AC005 root Roots 0.067 0.025 0.118 0 0 0 0 0.007 0.5 0.022 AC007seedling Seedling, 0.033 0.056 0.059 0.333 0 0 0 0 0 0.089 shoots androots AC008 flower Flowers, 0.033 0.087 0.059 0 0 0.038 0 0.007 0 0.022male and female organs AC009 shoot Cold shoots 0.067 0.193 0.059 0 00.075 0 0.028 0 0 (3, 6, 12, 24, 48) AC010 shoot Salt shoots 0 0.118 0 00 0.094 0 0.007 0 0.022 (6, 12, 24, 48 H) AC011 shoot Shoots 0 0.056 0 00 0.075 0 0.056 0 0 (2 + 8 H dark) AC012 root Salt roots 0.133 0.006 0 00 0 0 0.007 0 0 (6, 12, 24, 48 H) AC013 seed Seedlings, 0.033 0.043 0 00 0 0 0.014 0 0.111 seed and small shoot & root AC014 shoot Flooding0.033 0.012 0 0 0.333 0.019 0 0.084 0 0 shoots (5, 24, 48, 72 +24, 48 H) AC015 root Flooding 0.033 0.025 0 0 0.333 0.019 0.083 0.007 00 roots (5, 24, 48 + 24, 48 H) AC016 shoot Drought 0.133 0.031 0.059 0 00.019 0.417 0.308 0 0.044 shoots (24, 48 + 6, 12 H) AC018 root Drought 00.043 0 0.333 0 0.019 0.083 0 0 0.022 roots (24, 48 + 6, 12 H) AC019panicle Panicles 0 0.043 0 0 0.333 0 0 0 0 0.267 (pooled over 20 days)AC020 embryo Immature 0 0 0 0 0 0 0 0 0 0 embryos and endosperm AC021shoot Nipponbare 0 0.037 0 0 0 0.094 0 0.098 0 0 biotic stress 1 AC022flower Head flowers 0.1 0.012 0 0 0 0 0 0 0 0.022 (1-5, 10 15 Days)AC024 shoot Cypress 0.133 0.012 0.118 0 0 0.075 0 0.014 0 0 shoots AC025shoot Nipponbare 0.033 0.019 0.059 0 0 0.283 0.417 0.049 0 0.067biotic stress 3 AC026 shoot Nopponbare 0.1 0.037 0.059 0 0 0.038 0 0.0420 0 biotic stress 2 AC027 flower Cypress 0.033 0.012 0 0 0 0.038 0 0.0210 0.022 flowers AC092 RNA mix Combined 0 0.012 0 0 0 0 0 0.231 0 0mRNA long clone library Pre- Prevent Prevent Prevent Prevent Preventferred leakiness leakiness leakiness leakiness leakiness Use in every-in root and in shoot in shoot in shoot where but shoot and root uderand root embryo under flood drought condition and bacteria infectionTable 3-B (cont. from Table 3-A): miRNAs identified from Oryza sativa libraries.Os pri- miRNA ID Os 156^(a) Os miR160f Os miR162a Os miR164a Os miR164dOs miR166a Os UGACAGAAGA UGCCUGGCUC UCGAUAAACC UGGAGAAGCA UGGAGAAGCAUCGGACCAGG miRNA GAGUGAGCACA CCUGAAUGCCA UCUGCAUCCAG GGGCACGUGCAGGGCACGUGCU CUUCAUUCCCC sequence SEQ ID 6 9 10 11 12 13 NO: Hyseq35003089 35420108 39760468 34256080 34832815 35093513 clone ID LibraryLibrary Relative Relative Relative Relative Name Synonym DescriptionExpression Expression Expression Relative Expression Relative ExpressionExpression AC003 shoot Shoots 0 0 0 0 0 0 AC004 shoot Shoot meristems0.5 0 0.158 0.286 0 0.069 AC005 root Roots 0.5 0 0.158 0.143 0.333 0.241AC007 seedling Seedling, shoots 0 0 0 0 0 0 and roots AC008 flowerFlowers, male and 0 0.25 0.053 0 0 0 female organs AC009 shootCold shoots 0 0 0 0 0.333 0 (3, 6, 12, 24, 48) AC010 shoot Salt shoots 00 0 0 0 0 (6, 12, 24, 48 H) AC011 shoot Shoots (2 + 8 H dark) 0 0 0 0 00 AC012 root Salt roots 0 0 0 0.143 0 0.034 (6, 12, 24, 48 H) AC013 seedSeedlings, seed and 0 0 0.105 0 0 0.241 small shoot & root AC014 shootFlooding shoots 0 0 0.053 0 0 0 (5, 24, 48, 72 + 24, 48 H) AC015 rootFlooding roots 0 0 0 0 0 0.069 (5, 24, 48 + 24, 48 H) AC016 shootDrought shoots 0 0 0.053 0 0 0.138 (24, 48 + 6, 12 H) AC018 rootDrought roots 0 0 0 0 0 0 (24, 48 + 6, 12 H) AC019 paniclePanicles (pooled 0 0 0.263 0.286 0 0.103 over 20 days) AC020 embryoImmature embryos 0 0 0 0.143 0 0 and endosperm AC021 shootNipponbare biotic 0 0 0 0 0 0 stress 1 AC022 flower Head flowers (1-5, 00.125 0 0 0 0.034 10 15 Days) AC024 shoot Cypress shoots 0 0 0 0 0 0.034AC025 shoot Nipponbare biotic 0 0 0.053 0 0 0.034 stress 3 AC026 shootNopponbare biotic 0 0 0.053 0 0 0 stress 2 AC027 flower Cypress flowers0 0.625 0 0 0.333 0 AC092 RNA mix Combined mRNA 0 0 0.053 0 0 0long clone library Preferred Prevent leakiness Prevent leakinessPrevent leakiness Prevent leakiness Prevent leakiness Use in root andin flower in panicle in shoot tip in root, shoot shoot and shootand panicle (cold condition) and cypress flowers

TABLE 4 miRNAs identified from Zea mays sativa libraries.Zm pri-miRNA ID Zm miR156 Zm miR159 Zm miR160b Zm miR166 Zm miR167Zm miR171 ZmmiRNA sequence UGACAGA UUUGGAU UGCCUGGC UCGGACCAG UGAAGCUGUGAUUGAGC AGAGAGU UGAAGGG UCCCUGUA GCUUCAUUC CCAGCAUG CGCGCCAAU GAGCACAGCUCUA UGCCA CCC AUCUGG AUC SEQ ID NO: 22 23 24 25 26 27 Hyseq clone ID58989601 62202898 65442307 57507158 62178918 61430017 Library RelativeRelative Relative Relative Relative Relative Library Name SynonymDescription Expression Expression Expression Expression ExpressionExpression AC073 stem underground stem with 0.015 0.176 0.056 0 0 0meristem AC079 roots root only from young to 0 0 0 0.25 0 0mid-age plant AC080 tassel Tassel development 0 0 0 0 0 0 AC081 earEar development 0 0 0 0 0 0 AC082 Leaves Leaves of mixed ages, all 0.1230 0 0.25 0 0 prior to seed-fill AC083 immature Ear shoots from 2 cm0.031 0.059 0 0 0 0 ears (V13) up to and includingsilking (unpollinated). 51 to 70 dap. AC084 stem Stem tissue near ear at0 0 0 0 0 0 tassel emergence and during seed-fill AC085 uppper56 (pretasseling) and 84 0.015 0 0 0.5 0 0 leaves dap and 23 dpp (R3).Upper leaves at seed-fill AC086 seed Kernals at early dough 0 0 0 0 0.320 stage (R4). Developing starch grains and well- formed embryo present.30 d post pollination AC087 seed B73 inbreds. Kernals at 9 0 0 0 0 0 0and 19 d post pollination AC089 root Roots only from young to 0.062 0 00 0 0 mid-age plants AC088 seed Kernel at 9 and 19 d post 0.077 0.059 00 0 0 pollination AC093 stem Shoot cold, 10 d in chamber 0.062 0.4710.056 0 0 0.2 at 10 C./4 C. AC094 seed Very young kernels at 0 0 0 00.32 0 blister stage AC096 seed Mo17 inbreds. Kernals at 0 0 0.056 00.16 0.2 10 and 21 d post pollination AC095 seedKernals at early dent stage 0 0 0 0 0.08 0 (R5) AC099 RNA mixCombined mRNA long 0.031 0.235 0 0 0.08 0 clone library AC105 callusCallus from immature embros, 0.031 0 0 0 0 0infected with agrobacteriurm AC107 callus Normal callus from immature0.108 0 0.444 0 0 0 embryos at 7, 14, 31, 44, 65 d after cultivationAC113 shoot 3 sets: 1. Shoot, no water 0.246 0 0.111 0 0.04 0at V4 for 3, 7, 10 d +− 6 h recovery; 2. Shoot + root,dried 3, 6, 24 h +− 6 h water; 3. Shoot, no water at v15for 6, 9, 13, 16 d +/− 6 h water AC118 root 2 sets: 1. Root, no water 00 0.167 0 0 0 at V4 for 3, 7, 10 d +/− 6 h recovery; 2. root, air-dried3, 6, 24 h +/− 6 h rewater. AC120 root Roots only 0.169 0 0.111 0 0 0.2AC121 shoot Shoot only 0.031 0 0 0 0 0.4 Preferred use Prevent PreventPrevent leakiness Prevent leakiness Prevent leakiness leakinessleakiness in in leaves in in stem, everywhere stem and tassel seedsroot and shoot but seeds

TABLE 5 Mammalian miRNA, their miRNA-tags and their expression profilesSEQ ID NO miRNA Sequence Length of Skeletal miRNA ID (for miRNA)(miRNA Tag) miR-NA (nt) Brain Liver Heart muscle mmu-miR-1b 56Tag: TACATACTTCTTTACATTCCA 21 X X miRNA: UGGAAUGUAAAGAAGUAUGUA mmu-miR-957 Tag: TCATACAGCTAGATAACCAAAGA- 23 X miRNA: UCUUUGGUUAUCUAGCUGUAUGAmmu-miR-122a 58 Tag: ACAAACACCATTGTCACACTCCA 23 XmiRNA: UGGAGUGUGACAAUGGUGUUUGU mmu-miR-124a 59Tag: TGGCATTCACCGCGTGCCTTAA 22 X miRNA: UUAAGGCACGCGGUGAAUGCCAmmu-miR-128 60 Tag: AAAAGAGACCGGTTCACTGTGA 22 X XmiRNA: UCACAGUGAACCGGUCUCUUUU mmu-miR-194 61 Tag: TCCACATGGAGTTGCTGTTACA22 X miRNA: UGUAACAGCAACUCCAUGUGGA mmu-miR-206 62Tag: CCACACACTTCCTTACATTCCA 22 X X miRNA: UGGAAUGUAAGGAAGUGUGUGGhsa-miR-218 63 Tag: ACATGGTTAGATCAAGCACAA 21 XmiRNA: UUGUGCUUGAUCUAACCAUGU   Note: Northern analysis on human tissues(Ref: Sempere LF et al., Genome Biology 2004, R13)

TABLE 6 Maize miRNAs Identified in Hyseq Database Length Maize miRNAmiRNA ID SEQ ID NO: miRNA Sequence (nt) Precursors miR167-like 47UGAAGCUGCCAGCAUGAUCU 20 Contig9065 62178918ZM miR171 53UGAUUGAGCCGCGCCAAUAUC 21 Contig5235 61430017ZM miR167-like 48UGAAGCUGCCAGCAUGAUCUG 21 Contig9065 62178918ZM miR167 46UGAAGCUGCCAGCAUGAUCUGG 22 Contig9065 62178918ZM miR167-like 49UGAAGCUGCCAGCAUGAUCUAU 22 Contig9065 62178918ZM miR171-like 54UGAUUGAGCCGCGCCAAUAU 20 Contig5235 61430017ZM miR167-like 50AUGAAGCUGCCAGCAUGAUCUA 22 Contig9065 62178918ZM miR390 55AAGCUCAGGAGGGAUAGCGCC 21 Contig4340 59283967ZM miR167-like 51GUGAAGCUGCCAGCAUGAUCUA 22 Contig9065 62178918ZM miR166 42UCGGACCAGGCUUCAUUCCCC 21 57507158.f_k09_1 57507158.f_k09_1 miR156 31UGACAGAAGAGAGUGAGCAC 20 Contig3945 58989601ZM miR159 35UUUGGAUUGAAGGGAGCUCUA 21 Contig9470 62202898ZM miR160 39UGCCUGGCUCCCUGUAUGCCA 21 65442307.f_l16_1 65442307.f_l16_1 miR156-like32 UUGACAGAAGAGAGUGAGCAC 21 Contig3945 58989601ZM ASRP754-like 28AGCUCAGGAGGGAUAGCGCC 20 Contig4340 59283967ZM miR166-like-1 43UCGGACCAGGCUUCAUUCCCCC 22 57507158.f_k09_1 57507158.f_k09_1 miR159-like36 UUUGGAUUGAAGGGAGCUCUU 21 Contig9470 62202898ZM miR160-like 40UGCCUGGCUCCCUGUAUGCCAU 22 65442307.f_l16_1 65442307.f_l16_1 miR160-like41 GCCUGGCUCCCUGUAUGCCA 20 65442307.f_l16_1 65442307.f_l16_1 miR159-like37 UCUUUGGAUUGAAGGGAGCUC 21 Contig9470 62202898ZM miR166-like 44UUCGGACCAGGCUUCAUUCCCC 22 57507158.f_k09_1 57507158.f_k09_1 miR166-like45 UUCGGACCAGGCUUCAUUCCC 21 57507158.f_k09_1 57507158.f_k09_1miR156-like 33 GUGACAGAAGAGAGUGAGCAC 21 Contig3945 58989601ZMASRP754-like 29 AAGCUCAGGAGGGAUAGCGC 20 Contig4340 59283967ZMmiR159-like 38 UUUGGAUUGAAGGGAGCUCU 20 Contig9470 62202898ZM miR170-like52 UGAUUGAGCCGUGCCAAUAUC 21 58229137.f_c06_1 58229137.f_c06_1miR156-like 34 UGACAGAAGAGAGUGAGCACA 21 Contig3945 58989601ZMASRP754-like 30 AGCUCAGGAGGGAUAGCGCCA 21 Contig4340 59283967ZM

Example 4: Vector Construction for Plant Transformation

A typical plant transformation vector or binary vector contains twoplant expression constructs: one for selection marker and the other forgene-of-interest. Each cassette consists of a promoter, a gene to beexpressed and a terminator. The expression construct can be constructedinto a binary vector via standard molecular cloning procedures, PCR orvia Gateway system (Invitrogen, CA)

4.1 Isolation of Promoters

Genomic DNA from maize and rice is extracted using the Qiagen DNAeasyPlant Mini Kit (Qiagen). The promoter regions were isolated from genomicDNA using conventional PCR. Approximately 0.1 μg of digested genomic DNAwas uses for the regular PCR reaction (see below). The primers weredesigned based on the maize or rice genomic DNA sequences upstream ofthe EST candidates, maize genomic sequences, or promoter sequencesdisclosed in the public database (e.g. rice caffeoylCoA-O-methyltransferase [CCoAMT1], GenBank accession number AB023482;rice unknown protein, AP002818; maize hydroxyproline-rich glycoprotein[HRGP], AJ131535; maize lactate dehydrogenase [LDH], Z11754; riceChloroplast Protein12-like, NP914106.1). 1 μL of the diluted digestedgenomic DNA was used as the DNA template in the primary PCR reaction.The reaction comprised forward (5′) and reverse (3′) primers in amixture containing Buffer 3 following the protocol outlined by an ExpandLong PCR kit (Cat #1681-842, Roche-Boehringer Mannheim). The isolatedDNA is employed as template DNA in a PCR amplification reaction usingthe following primers:

TABLE 7 Primer sequences for isolation of the promoter regionPromoter or Size Primer Sequences Terminator* (bp)Forward Primer (F) & Reverse Primer (R) Oryza sativa 1,035F: 5′-CAACTACTGCACGGTAAAAGTGATAGG-3′ Caffeoyl-CoA-O- (SEQ ID NO: 64)methyltransferase R: 5′-GCAGCTTGCTTCGATCTCTCGCTCGCC-3′ Promoter(SEQ ID NO: 65) (Os.CCoAMT1-p) Oryza sativa 813FP: 5′-TGCCTCGATTCGACCGTGTAATGGAAT-3′ C-8,7-sterol-isomerase(SEQ ID NO: 66) Promoter RP: 5′-ACTCCTGGCTTCCTTCCGATCTGGACT-3′ (Os.SI-p)(SEQ ID NO: 67) Zea maize 1,263 FP: 5′-CCGGTGACCTTCTTGCTTCTTCGATCG-3′Hydroxyproline-rich (SEQ ID NO: 68) glycoprotein PromoterRP: 5′-CCTCTCTCTCACACACACTCTCAGTAA-3′ (Zm.HRGP-p) (SEQ ID NO: 69)Zea maize 1,061 FP: 5′-AACAAATGGCGTACTTATATAACCACA-3′Lactate-dehydrogenase (SEQ ID NO: 70) promoterRP: 5′-CGGGCGGAATGGGATGGGATTACGTGT-3′ (Zm.LDH-p) (SEQ ID NO: 71)Oryza sativa 998 FP: 5′-TTTGTATTTAGGTCCCTAACGCCCTC-3′Chloroplast protein 12 (SEQ ID NO: #72) PromoterRP: 5′-TGTTGATGCGGATTTCTGCGTGTGAT-3′ (Os.CP12-p) (SEQ ID NO: 73)

The promoter regions are amplified in the reaction solution[1×PCRreaction buffer (Roche Diagnostics), 5 μL genomic DNA (corresponds toapproximately 80 ng, 2.5 mM of each dATP, dCTP, dGTP and dTTP(Invitrogen: dNTP mix), 1 μL 5′ primer (100 μM) 1 μL 3′ primer (100 μM),1 μL Taq DNA polymerase 5 U/μL (Roche Diagnostics), in a final volume of100 μL] under the optimized PCR thermocycler program (T3 ThermocyclerBiometra; 1 cycle with 180 sec at 95° C., 30 cycles with 40 sec at 95°C., 60 sec at 53° C. and 2 min at 72° C., and 1 cycle with 5 min at 72°C. before stop the reaction at 4° C.).

The PCR product was applied to a 1% (w/v) agarose gel and separated at80V followed by excising from the gel and purified with the aid of theQiagen Gel Extraction Kit (Qiagen, Hilden, Germany). If appropriate, theeluate of 50 μL can be evaporated. The PCR product was cloned directlyinto vector pCR4-TOPO (Invitrogen) following the manufacturer'sinstructions, i.e. the PCR product obtained is inserted into a vectorhaving T overhangs with its A overhangs and a topoisomerase.

4.2 Isolation of Terminator of Interest Including the 3′ UntranslatedRegion

Genomic DNA fragment containing the 3′ untranslated regions of interestwere isolated using sequence specific primers based on the sequencesthat disclosed in the public database (GenBank accession numberAB023482, AJ131535, Z11754; Table 8). Plant genomic DNA isolation andconventional PCR amplification using sequence specific primers wereconducted using the protocols in the art (Sambrook, 1987).

TABLE 8 Primer sequences for isolation of terminator region SizePrimer Sequences Terminator (bp) Forward Primer (F) & Reverse Primer (R)Oryza sativa 1,092 FP: 5′-GCCGATGCCCAAGAACTAGTCATTTTA-3′ Caffeoyl-CoA-O-(SEQ ID NO: 74) methyltransferase RP: 5′-ATTAACACGTCAACCAAACCGCCGTCC-3′Terminator (SEQ ID NO: 75) (Os.CCoAMT1-t) Zea maize 541FP: 5′-AAAGCGATGCCTACCATACCACACTGC-3′ Hydroxyproline-rich glyco-(SEQ ID NO: 76) protein TerminatorRP: 5′-TGCCCACATTTATTATGGTTTTACACCC-3′ (Zm.HRGP-t) (SEQ ID NO: 77)Zea maize 475 FP: 5′-TGATCACATCACCGTCTCTCTTCATTAA-3′Lactate-dehydrogenase (SEQ ID NO: 78) terminatorRP: 5′-TATCCCAGTCTCGATATTGTCATCCGCT-3′ (Zm.LDH-t) (SEQ ID NO: 79)

The primer sequences given in the table abobe represent the 3′-part ofthe actual primer used. Said primers further comprised aSacI-restriction site adapter (5′-GAGCTC-3′) for the forward primer anda PmeI-resitrtiction site adapter (5′-GTTTAAAC-3′) for the reverseprimer (added to the sequence-specific primers for the further cloningpurpose.

4.3 pUC Based Vector (Promoter of Interest::Intron(IME)::GUS::Terminator)

The base vector (pBPSMM270) comprises multiple cloning sites (MCS)followed by the first Zm.ubiquitin intron, the GUSint ORF (including thepotato invertase [PIV]2 intron to prevent bacterial expression), andnopaline synthase (NOS) terminator in order (5′ to 3′). Maize ubiquitinintron can be replaced with an intron of interest that functions inintronmeidated enhancement at BgII and XmaI.

The genomic DNA fragment containing promoter of interest (Os.CCoAMT1,Os.SI, Zm.HRGP, Zm.LDH, or Os.CP12 promoter) in the Topo vector(Invitrogen) was digested with PacI and AscI followed by subcloningupstream of the Zm.ubiquitin intron into the GUS construct.

The PCR fragment containing terminator of interest (e.g. 1,092 bp ricegenomic DNA including CCoAMT1 terminator; 558 bp maize genomic DNAincluding HRGP terminator, 477 bp maize genomic DNA including LDHterminator) was digested with SacI and PmeI enzymes. Nopaline synthaseterminator region was replaced with terminator of interest.

In order to include a miRNA tag in the terminator region, thecomplementary sequences (up to 21 bp) of the miRNAs of interest (Table9) are chemically synthesized including a SacI restriction enzyme siteat both 5′ and 3′-ends of the sequence followed by suncloning betweenGUS gene and terminator of interest. The tag can be inserted into 5′UTRor 3′UTG or the coding region of gene-of-interest without affecting genefunction.

TABLE 9 BPS miRNA tag sequences and the expression patterns miRNA tagSEQ Tag miRNA [MRT] ID ID NO sequence (5′ to 3′) expression BPS.MRT1 80ACAGATCATGCTGGCAGCTTCA Predominantly in seed BPS.MRT2 81TAGAGCTCCCTTCAATCCAAA Non-seed tissues

4.4 Transformation Binary Vector (Promoter Constructs without a miRNATag)

The GUS chimeric cassettes in the pUC-based vectors were digested withAscI or PacI (5′) and PmeI (3′) and subcloned into a monocot binaryvector containing a plant selectable marker cassette (pBPSMM344) at AscIor PacI (5′) and PmlI (3′) sites to generate promoter constructs forplant transformation (Table 10).

TABLE 10 Promoter constructs without a miRNA tag in a binary vectorPromoter SEQ ID Composition [promoter (p) ::IME-intron (i) ::GUS::terminator (t)] construct NO: (Numbers are indicating the nucleotideposition in the construct) pBPSMM232 84Zm.ubiquitin-p::Zm.ubiquitin-i::GUS (PIV2)::NOS-t Zm ubiquitin promoterand intron (1981 bp): 298-2278, GUS (2001 bp): 2305-4305, NOS terminator(253 bp): 4376-4628 MiRNA tag insertion site: 4365 (Sac I) pBPSMM271 85Os.CCoAMT1-p::Zm.ubiquitin-i::GUS (PIV2)::NOS-t Os CCoAMT1 promoter(1034 bp): 227-1260, Zm ubiquitin intron (1051 bp): 1319-2369, GUS (2001bp): 2389-4389, NOS terminator (253 bp).: 4461-4713 MiRNA tag insertionsite: 4450 (Sac I) pBPSMM272 86 Zm.LDH-p::Zm.ubiquitin-i::GUS(PIV2)::NOS-t Zm LDH Promoter (1062 bp): 255-1316, ubiquitin Intron(1051 bp): 1355-2405, GUS (2001 bp): 2425-4425, NOS terminator (253 bp):4497-4749 MiRNA tag insertion site: 4486 (SacI) PBPSMM304 87Os.CP12-p::Zm.ubiquitin-i::GUS (PIV2)::NOS-t Os CP12 promoter (998 bp):3859-4856, Zm ubiquitin intron (1051 bp): 2769-3819, GUS (2001 bp):749-2749, NOS terminator (253 bp): 426-678 MiRNA tag insertion site: 694(Sac I) pBPSMM331 88 Os.SI-p::Zm.ubiquitin-i::GUS (PIV2)::NOS-t Os SIpromoter (814 bp): 3912-4725, Zm ubiquitin intron (1051 bp): 2824-3874,GUS (2001 bp): 804-2804, NOS terminator (253 bp): 481-733 MiRNA taginsertion site: 749 (Sac I) pBPSMM325 89Os.CCoAMT1-p::Zm.ubiquitin-i:GUS (PIV2)::CCoAMT1-t Os CCoAMT1 promoter(1034 bp): 305-1338, Zm ubiquitin intron: (1051 bp) 1345-2395, GUS (2001bp): 2407-4407, CCoAMT1 terminator (1104 bp): 4446-5549. MiRNA taginsertion: 4793 (AgeI) pBPSET003 Zm.HRGP-p::Zm.ubiquitin-i::GUS(PIV2)::Zm.HRGP-t pBPSET007 Zm.LDH-p::Zm.ubiquitin-i::GUS(PIV2)::Zm.LDH-t

The promoters described above are preferably improvement for higherpromoter specificity with miRNA tags as follows: The Os CCoAMT promoterwith ubiquitin intron (pBPSMM271) or the Zm LDH promoter with ubiquitinintron (pBPSMM272) are active strongly in roots and kernel. By use of aZm miR167 tag, the expression in kernel is eliminated andgene-of-interest becomes predominantly expressed in roots.

The Os CP12 promoter with ubiquitin intron is active strongly in leafs,but medium in endosperm, no activity is observed in roots and embryos.By use of Zm miR167 tag, the expression in endosperm is eliminated andgene-of-interest becomes expressed predominantly in leaves.Alternatively, by use of a Zm miR166h tag, expression in leaves isreduced or eliminated and gene-of-interest is expressed predominantly inendosperm.

The Os SI promoter with ubiquitin intron is active strongly in roots andkernel, but weakly in leaves. By use of a Zm miR166h tag, expression inleaves is eliminated and the gene-of-interest is predominantly expressedin roots.

A miRNA tag can be introduced into the beginning of the terminator afterthe stop codon of GUS gene using PCR and standard cloning methods. Forexample, the insertion can be realized by utilizing a unique SacI site(Sac I and Pac I are unique sites to remove NOS terminator frompBPSMM271). The miRNA-tag for miR166h is incorporated into NOSterminator region by PCR with following primers:

Forward primer (SEQ ID NO: 209): 5′-GGGAGCTCGGGGAATGAAGCCTGGTCCGAgaatttccccgatcg ttcaaacatttggca(The SacI-site is underlined; The miR166h-tag is in bold letters.Reverse primer (SEQ ID NO: 210): 5′ TCGGACCGTTAATTAACACAAACTGAAGGCThe Pac I site is underlined.

The template DNA is the vector prior to insertion of the miRNA-tag. ThePCR product is subsequently cut with restriction enzyme Sac I and Pac I.This fragment is then ‘swaped’ with Sac I-Pac I fragment in MM271 bysubcloning. This strategy can be used to engineer miRNA tags into NOSterminator for other binary vectors such as pBPS MM 272, MM232 andMM304.

Example 5: Engineering Binary Vector with Tissue-Specific miRNA Tags toTarget DsRed mRNA

A binary vector, pBPSLM185 (SEQ ID NO: 212), contains a reporter geneexpression construct: ScBV promoter (1398 bp), a full-length DsRed cDNA(678 bp) and a NOS terminator (253 bp). ScBV promoter was isolated fromsugarcane bacilliform badnavirus (Schenk et al., Plant Mol Biol.39:1221-1230, (2004))). DsRed is red fluorescent protein from Discosomasp. reef coral (Baird, G. S., et al., Proc. Natl. Acas. Sci USA97:11984-11989, (2000)). NOS terminator is 3′ untranslated region ofnopaline synthase gene isolated from Agrobacterium. In transgenic maizecarrying LM185 construct, strong red fluorescence was readily detectedthrough out plants by fluorescence microscopy analysis. The quantitativeanalysis of expression of DsRed was achieved by using an imaginginstrument (Typhoon 9400, Amersham Biosciences). The ubiquitousexpression of DsRed was resulted from ScBV promoter which is active inevery maize tissues.

To reduce or eliminate expression of DsRed in maize leaf and tassel, amodified LM185 binary vector, PR100, is constructed. In vector PR100, ashort nucleotide sequence or lag′ is cloned into NOS terminator regionby PCR using LM185 DNA as a template.

Forward primer (SEQ ID NO: 209): 5′-GGGAGCTCGGGGAATGAAGCCTGGTCCGAgaatttccccgatcg ttcaaacatttggca(The SacI-site is underlined; The miR166h-tag is in bold letters.Reverse primer (SEQ ID NO: 211): 5′ GATCTGGCCGGCCGGGCCCGAATTCThe FseI site is underlined.

The PCR product is subsequently cut with restriction enzyme Sac I andFseI. This fragment is then ‘swaped’ with Sac I-FseI fragment in LM185by subcloning. The resulted PCR product contains ‘tag 1’ at thebeginning of NOS terminator. The restriction sites at each end of PCRproduct facilitates subcloning such modified NOS terminator into binaryvector following DsRed coding sequence. This strategy applies tointroduce any miRNA tags into expression cassette. The ‘tag 1’ sequence,5′ GGGGAATGAAGCCTGGTCCGA 3′ (SEQ ID NO: 82) is completely complementaryto maize miRNA miR166h, 5′ TCGGACCAGGCTTCATTCCCC 3′. Transgenic maizecarrying PR100 express DsRed mRNA with a ‘tag 1’ in every maize tissue.Because miR166h is only expressed in leaves and tassels, miR166hrecognizes and binds to the lag′ specifically in DsRed mRNA in theleaves and tassels. As a result, DsRed mRNA levels in these tissues arereduced or eliminated through miRNA-mediated gene silencing. The redfluorescence is reduced or undetectable in leaves and tassels but is notaffected in other tissues.

It has been shown both in animal and plant, complementarity between 5′region of miRNA (e.g. position 2-8 nt) and miRNA target site is crucialfor miRNA action (Mallory et al., EMBO Journal, 23:3356-3364, (2004),Doench J and Sharp P, Genes & Development 504-511, (2004)), while 3′region of miRNA (e.g. position 12-19 nt) can be mismatched to its targetsite. Such mismatch might reduce the efficacy of miRNA-mediated genesilencing.

A binary vector, PR101, is the same as PR100 except the ‘tag 2’ is usedinstead of ‘tag 1’. The ‘tag 2’, 5′ GGGGAATGAAGCgTGGaCCGA 3′ (SEQ ID NO:82) contains two mutations ‘C to g’ and ‘T to a’ comparing to ‘tag 1’.This results in two mismatches between lag 2′ and miR166h. Transgenicmaize carrying PR101 has reduced red fluorescence in leaves and tassels.Furthermore, quantitative analysis on multiple events of transgenicmaize carrying PR100 or PR101 using an imaging instrument (e.g. Typhoon9400) shows that statistically the intensity of red fluorescence fromPR100 maize is lower than that from PR101 maize in leaves and tassels.This is because perfect complementarity between ‘tag 1’ and miR166h inPR100 causing great reduction of DsRed expression, whereas mismatchesbetween ‘tag 2’ and miR166h in PR101 causing less reduction of DsRedexpression in leaves and tassels.

Example 6: Engineering Binary Vector with Tissue-Specific miRNA Tags toTarget a Trait Gene or Selection Marker

Seeds are the most relevant agronomical product, which is heavily usedfor feed and food purposes. However, expression of transgenes in seedsis in most cases neither necessary nor beneficial. For example, traitslike herbicide resistance, resistance against insects, fungi, ornematode, cold or drought resistance do not need to be expressed inseeds, since expression is only required in roots or green tissues.Expression in seeds can have one or more of the followingdisadvantageous:

-   1. Unnecessary expression of traits in seeds may lead to lower    germination rates or at least unnecessary consumption of    transcription/translation capacity resulting in yield loss or    negatively affecting composition of the seed.-   2. Unnecessary expression of traits in seeds may raise higher    hurdles in de-regulation proceedings (since a more substantial    amount of the transgenic product is comprised in the feed or food    materials).-   3. Unnecessary expression of traits in seeds may negatively affect    consumer acceptance.

Flowers comprise the plants reproductive organs (carpels and stamens).Expression in these tissues is for some traits also regarded asdisadvantageous. For example, expression of the Bt protein (conferringresistance against corn root borer and other insect pests) under astrong constitutive promoter resulted in expression in pollen and wasdiscussed to have a toxic effect on beneficial pollen transferringinsects like the monarch butterflies.

A point mutation of a single nucleotide in AHAS (acetohydroxyacidsynthase) gene generates resistance to herbicide imidazolinone. Amutated version of AHAS is also used as a selection marker for croptransformation towards commercial application. To eliminate AHAS markerin the seeds, a binary vector carrying miR-167 tag can be constructed.Maize miR167 is predominantly expressed in seeds including differentstages of seed development. In a binary vector PR102, Ubi promoterdrives AHAS expression. Following AHAS cDNA, a short nucleotide sequenceor ‘tag’ is cloned into NOS terminator by PCR and standard cloningprocedure. The ‘tag 3’ sequence, 5′ ACAGATCATGCTGGCAGCTTCA 3′ (SEQ IDNO: 80) is completely complementary to maize miRNA miR167, 5′TGAAGCTGCCAGCATGATCTGT 3′. Transgenic maize carrying PR102 express AHASmRNA with a ‘tag 3’ in every maize tissue. Because miR167 ispredominantly expressed in seeds, miR167 recognizes and binds to the‘tag’ specifically in AHAS mRNA in the seeds. As a result, AHAS mRNAlevels in seeds is reduced or eliminated through miRNA-mediated genesilencing. The AHAS expression in other tissues is largely unaffecteddetermined by Western blot analysis using an antibody specificallyrecognize a mutated AHAS.

Example 7: From Constitutive Expression to Vegetative Tissue-Specific orKernel-Specific Expression

7.1 Constitutive Expression [without a miRNA Tag]

In comparison with maize ubiquitin promoter (Zm.ubiquitinpromoter::Zm.ubiquitin intron) and sugarcane bacilliform virus promoter(pBPSMM247), rice CCoAMT1 promoter in combination with Zm.ubiquitinintron and CCoAMT1 terminator (pBPSMM325) showed medium to strongconstitutive and ubiquitous expression in all tissues and organs atdifferent developmental stages. Strong ubiquitous expression can also bedetected in in vitro plants.

TABLE 11 GUS expression controlled by monocot constitutive promotercandidates Promoter (GUS expression levels) Tissues/Developmental stagespBPSMM232* pBPSMM247* pBPSMM325 3 days after co-cultivation ++++ +++ +++Leaves at 5-leaf stage +++++ +++++ ++++ Roots at 5-leaf stage ++++++++++ ++++ Leaves at flowering stage +++++ +++++ +++ Stem +++ +++ +++Pre-pollination +++++ +++++ ++ 5 days after pollination [DAP] +++++ +++(7 DAP) ND 30 DAP +++++ +++++ ++ Dry seeds ND +++ ++Imbibition/germination +++++ ++++ ND *Positive controls as aconstitutive promoter (pBPSMM232 = Zm.ubiquitin promoter::Zm.ubiquitinintron::GUS (PIV2)::NOS terminator; pBPSMM247 = sugarcane bacilliformvirus promoter::GUS (PIV2) ::NOS terminator); pBPSMM325 = Os.CCoAMT1promoter::Zm.ubiquitin intron::GUS (PIV)2::Os.CCoAMT1 terminator; arange of GUS expression levels measured by histochemical assay (− to+++++), ND: not determined yet

7.2 Vegetative Tissue-Specific or Kernel-Specific Expression Controlledby miRNA Tag in the Terminator Region

To control either vegetative tissue-specific or kernel-specificexpression, BPS.MRT1 or BPS.MRT2 is inserted between GUS gene and NOSterminator at SacI site in pBPSMM232, pBPSMM247, or pBPSMM235 togenerate pBPSPR1 or pBPSPR002, pBPSPR003 or pBPSPR004, or pBPSPR005 orpBPSPR006, respectively. A chimeric construct composed of a miRNA tagcan be transformed into monocotyledonous or dicotyledonous plants suchas rice, barley, maize, wheat, ryegrass, Arabidoposis, canola, soybean,tobacco, but is not restricted to these plant species. Any methods forimproving expression in monocotyledonous plants are applicable such asaddition of intron or exon with intron in 5′UTR either non-spliced orspliced. Standard methods for transformation in the art can be used ifrequired. Transformed plants are selected under the selection agent ofinterest and regenerated using known methods. Selection scheme isexamined at early developmental stages of tissues or tissue culturecells. Gene expression levels can be determined at different stages ofdevelopment and at different generations (T0 to T2 plants or furthergenerations). Results of the evaluation in plants lead to determineappropriate genes to be used in this promoter construct.

Example 8: From Root and Kernel-Preferable Expression to Root orKernel-Specific Expression

8.1 Root and Kernel-Preferable Expression [without a miRNA Tag]

The following four promoter constructs showed root and kernel-preferableexpression in maize (Table 12). First, riceCaffeoyl-CoA-O-methyltransferase (CCoAMT1)promoter::ubiquitin-intron::NOS terminator (pBPSMM271) showed lowexpression in leaves and stem of T1 plants but strong expression inroots. GUS stain was also detected in kernel and pollen.

Second, OsC-8,7-sterol-isomerase promoter::Zm.ubiquitinintron::NOSterminator (pBPSMM331) showed weak expression in most parts of theplants but good expression in roots and kernels. Third, maize HRGPpromoter containing the ubiquitin intron and the HRGP termiantor(pBPSET003) showed no expression in leaves but strong expression inroots and silk. In kernels expression is predominantly in the embryo andonly weak in the endosperm. Fourth, maize Lactate-dehydrogenase (LDH)promoter::Zm.ubiquitinintron::NOS or LDH terminator (pBPSMM272 orpBPSET007, respectively) showed weak expression in leaves but goodexpression in roots and kernels.

TABLE 12 GUS expression controlled by monocot root and kernel-preferablepromoter candidates Promoter (GUS expression levels) pBPSMM272 Tissues &or Developmental stages pBPSMM232* pBPSMM271 pBPSMM331 pBPSET003pBPSET007 3 days after co- ++++ + ND ND +++ cultivation Leaves at 5-leafstage +++++ + + − ++ Roots at 5-leaf stage +++++ ++++ +++ ++++ ++++Leaves at flowering stage +++++ + ++ − ++ Stem +++ + ND ND +Pre-pollination +++++ +++ ++++ ND +++ 5 days after +++++ +++ ND ND +++pollination [DAP] 30 DAP ++++ +++ ++ ++ +++ Dry seeds ND ND ND ND NDImbibition/germination +++++ +++ ND ND +++ *positive control as aconstitutive promoter (pBPSMM232 = Zm.ubiquitin promoter::Zm.ubiquitinintron::GUS (PIV2)::NOS terminator); a range of GUS expression levelsmeasured by histochemical assay (− to +++++), ND: not determined yet

8.2 Root or Kernel-Specific Expression Controlled by miRNA Tag in theTerminator Region

To control either root-specific or kernel-specific expression, BPS.MRT1or BPS.MRT2 is inserted between GUS gene and NOS terminator at SacI sitein pBPSMM271, pBPSMM272, pBPSMM331, pBPSET003, or pBPSET007 to generatepBPSPR007 or pBPSPR008, pBPRPR009 or pBPSPR010, pBPRPR011 or pBPSPR012,pBPRPR013 or pBPSPR014, or pBPRPR015 or pBPSPR016, respectively. Achimeric construct composed of a miRNA tag can be transformed intomonocotyledonous or dicotyledonous plants such as rice, barley, maize,wheat, ryegrass, Arabidoposis, canola, soybean, tobacco, but is notrestricted to these plant species. Any methods for improving expressionin monocotyledonous plants are applicable such as addition of intron orexon with intron in 5′UTR either non-spliced or spliced. Standardmethods for transformation in the art can be used if required.Transformed plants are selected under the selection agent of interestand regenerated using known methods. Selection scheme is examined atearly developmental stages of tissues or tissue culture cells. Geneexpression levels can be determined at different stages of developmentand at different generations (T0 to T2 plants or further generations).Results of the evaluation in plants lead to determine appropriate genesto be used in this promoter construct.

Example 9: From Leaf and Endoperm-Preferable Expression to Leaf-Specificor Endosperm-Specific Expression

9.1 Leaf and Endosperm-Preferable Expression [without a miRNA Tag]

Os.CP12 promoter::Zm.ubiquitin intron::GUS (PIV2)::NOS terminator(pBPSMM304) showed strong expression in leaves and endosperm, but not inroots or embryo.

TABLE 13 GUS expression controlled by leaf and endosperm-preferablemonocot promoter Promoter (GUS expression levels) Tissues/Developmentalstages pBPSMM232* pBPSMM304 3 days after co-cultivation ++++ + In vitroleaves +++++ ++++ In vitro roots +++++ − Leaves +++++ ++++ Roots +++++ −Kernel pre-pollination +++++ + Kernel 30 DAP - Endosperm +++++ ++++Kernel 30 DAP - Embryo +++++ − Dry seeds ++++ ND *positive control as aconstitutive promoter (pBPSMM232 = Zm.ubiquitin promoter::Zm.ubiquitinintron::GUS (PIV2)::NOS terminator); a range of GUS expression levelsmeasured by histochemical assay (− to +++++), ND: not determined yet

9.2 Leaf or Endosperm-Specific Expression Controlled by miRNA Tag in theTerminator Region

To control either leaf-specific or endosperm-specific expression,BPS.MRT1 or BPS.MRT2 is inserted between GUS gene and NOS terminator atSacI site in pBPS304 to generate pBPSPR017 or pBPSPR018, respectively. Achimeric construct composed of a miRNA tag can be transformed intomonocotyledonous or dicotyledonous plants such as rice, barley, maize,wheat, ryegrass, Arabidoposis, canola, soybean, tobacco, but is notrestricted to these plant species. Any methods for improving expressionin monocotyledonous plants are applicable such as addition of intron orexon with intron in 5′UTR either non-spliced or spliced. Standardmethods for transformation in the art can be used if required.Transformed plants are selected under the selection agent of interestand regenerated using known methods. Selection scheme is examined atearly developmental stages of tissues or tissue culture cells. Geneexpression levels can be determined at different stages of developmentand at different generations (T0 to T2 plants or further generations).Results of the evaluation in plants lead to determine appropriate genesto be used in this promoter construct.

Example 10: Mature microRNA Profiling

Expression profiling of mature miRNAs in maize tissues was obtainedusing the 46 Arabidopsis thaliana (Ath) miRNA assays developed byApplied Biosystems (Chen et al. 2005, Nucleic Acids Research. 33:e179).Table 14 represents the 46 miRNA sequences that were used for theprofiling.

Total RNA from 11 different maize samples (Table 14) was extracted withTrizol reagent following the instruction recommended by manufactory(Invitrogen 15596-026). Maize glyceraldehyde-3-phosphate dehydrogenase(GADPH) subunit C was used for an internal control to normalize miRNAexpression among different tissue samples.

TABLE 14 miRNA sequence for the profiling Name Sequence SEQ ID NOAth-miR156 UGACAGAAGAGAGUGAGCAC 225 Ath-miR156g CGACAGAAGAGAGUGAGCACA226 Ath-miR156h UUGACAGAAGAAAGAGAGCAC 227 Ath-miR157UUGACAGAAGAUAGAGAGCAC 228 Ath-miR158 UCCCAAAUGUAGACAAAGCA 229Ath-miR159a UUUGGAUUGAAGGGAGCUCUA 230 Ath-miR159b UUUGGAUUGAAGGGAGCUCUU231 Ath-miR159c UUUGGAUUGAAGGGAGCUCCU 232 Ath-miR160UGCCUGGCUCCCUGUAUGCCA 1 Ath-miR161 UUGAAAGUGACUACAUCGGGG 233 Ath-miR162UCGAUAAACCUCUGCAUCCAG 234 Ath-miR163 UUGAAGAGGACUUGGAACUUCGAU 2Ath-miR164 UGGAGAAGCAGGGCACGUGCA 235 Ath-miR164c UGGAGAAGCAGGGCACGUGCG236 Ath-miR165 UCGGACCAGGCUUCAUCCCCC 237 Ath-miR166UCGGACCAGGCUUCAUUCCCC 238 Ath-miR167 UGAAGCUGCCAGCAUGAUCUA 3 Ath-miR167cUUAAGCUGCCAGCAUGAUCUU 239 Ath-miR167d UGAAGCUGCCAGCAUGAUCUGG 240Ath-miR168 UCGCUUGGUGCAGGUCGGGAA 241 Ath-miR169 CAGCCAAGGAUGACUUGCCGA242 Ath-miR169b CAGCCAAGGAUGACUUGCCGG 243 Ath-miR169dUGAGCCAAGGAUGACUUGCCG 244 Ath-miR169h UAGCCAAGGAUGACUUGCCUG 245Ath-miR170 UGAUUGAGCCGUGUCAAUAUC 246 Ath-miR171 UGAUUGAGCCGCGCCAAUAUC247 Ath-miR171b UUGAGCCGUGCCAAUAUCACG 248 Ath-miR172AGAAUCUUGAUGAUGCUGCAU 4 Ath-miR173 UUCGCUUGCAGAGAGAAAUCAC 249 Ath-miR319UUGGACUGAAGGGAGCUCCC 250 Ath-miR319c UUGGACUGAAGGGAGCUCCU 251Ath-miR393a UCCAAAGGGAUCGCAUUGAUC 252 Ath-miR394a UUGGCAUUCUGUCCACCUCC253 Ath-miR395a CUGAAGUGUUUGGGGGAACUC 254 Ath-miR395bCUGAAGUGUUUGGGGGGACUC 255 Ath-miR396a UUCCACAGCUUUCUUGAACUG 256Ath-miR396b UUCCACAGCUUUCUUGAACUU 257 Ath-miR397a UCAUUGAGUGCAGCGUUGAUG258 Ath-miR397b UCAUUGAGUGCAUCGUUGAUG 259 Ath-miR398aUGUGUUCUCAGGUCACCCCUU 260 Ath-miR398b UGUGUUCUCAGGUCACCCCUG 261Ath-miR399a UGCCAAAGGAGAUUUGCCCUG 262 Ath-miR399b UGCCAAAGGAGAGUUGCCCUG263 Ath-miR399d UGCCAAAGGAGAUUUGCCCCG 264 Ath-miR399eUGCCAAAGGAGAUUUGCCUCG 265 Ath-miR399f UGCCAAAGGAGAUUUGCCCGG 266

TABLE 15 Maize materials used for miRNA profiling Library ID TissueDescription AC094 Kernel 16 days post pollination. Kernels at blisterstage R2 AC081 Kernel 23 days post pollination. Kernels only (milkstage) AC086 Kernel 30 days post pollination. Kernels at R4, early doughAC095 Kernel 36 days post pollination. Kernels at beginning of dentstage, early R5 AC089 Root Roots (only), 2 leaf to 9 leaf stages. Fromgreenhouse plants, 12 dap (V2), 21 dap (V6), and 35 dap (V9). AC118 RootTwo samples were combined for the drought maize root library AC085 Upperleaf 56 (pretasseling) and 84 dap and 23 dpp (R3). Upper leaves atseed-fill AC082 Lower leaf Lower leaf tissue, from 12 dap (V2), 21 dap(V6), and 56 dap (pretassel). AC080 Ear 1 and 9 days post pollinationAC079 Tassel Immature and mature tassels at 44, 51, 56, 70dap(anthesis). Stages are 10-leaf, 13-leaf, just before tasselemergence, and anthesis (V10 to R1). Callus 21 days Severaltissue-specific miRNAs were identified through the profiling (Table 16).

TABLE 16 Relative expression of miRNAs in different maize tissues.Average of relative ex- lower upper root, kernel, kernel, kernel,kernel, pression level leaf leaf root drought tassel R2 R3 R4 R5 earcallus ath-miR156 0.05 0.06 36.29 0.36 9.72 0.12 1469.50 996.49 15.550.00 134.96 ath-miR164 0.62 0.14 39.69 2.61 9.09 0.84 345.50 289.53 4.271.61 2.01 ath-miR170 0.02 0.01 8.20 0.07 2.51 0.60 2112.79 1719.49 14.360.02 1.47 ath-miR396b 0.00 0.00 0.11 0.00 0.95 0.00 142.89 40.11 0.100.00 0.26 ath-miR156g 0.02 0.04 21.23 0.14 3.79 0.04 751.31 544.26 5.820.00 65.12 ath-miR164c 0.22 0.08 17.68 1.09 5.39 0.34 406.60 128.18 1.820.50 0.52 ath-miR171 0.01 0.00 6.23 0.06 2.46 0.46 2024.72 1644.35 4.130.01 2.32 ath-miR397a 0.00 0.00 0.00 0.00 0.05 0.00 59.70 19.52 0.040.00 3.93 ath-miR156h 0.00 0.00 0.08 0.00 0.02 0.00 2.96 3.23 0.02 0.000.00 ath-miR165 0.45 0.39 15.80 2.29 4.09 0.70 3660.80 1740.77 15.340.44 0.00 ath-miR171b 0.00 0.01 0.43 0.02 1.99 0.02 276.84 54.19 0.240.00 0.00 ath-miR397b 0.00 0.00 0.00 0.00 0.00 0.00 0.14 0.11 0.00 0.000.00 ath-miR157 0.00 0.00 0.04 0.00 0.02 0.00 1.62 1.58 0.02 0.00 0.10ath-miR166 0.52 0.84 27.81 5.26 10.15 1.05 7512.02 3237.04 37.50 0.7932.76 ath-miR172 0.00 0.04 30.39 0.05 6.89 0.01 610.71 138.30 0.23 0.000.22 ath-miR398a 0.03 0.10 0.03 0.02 0.17 0.16 139.69 31.63 0.30 0.000.81 ath-miR158 0.00 0.00 0.00 0.00 0.00 0.00 0.03 0.02 0.00 0.00 0.00ath-miR167 0.24 0.49 52.18 2.42 16.13 2.95 19579.50 15335.46 83.98 0.4492.18 ath-miR173 0.00 0.00 0.00 0.00 0.00 0.00 0.03 0.02 0.00 0.00 0.00ath-miR398b 0.07 0.12 0.02 0.02 0.18 0.15 113.83 24.07 0.45 0.00 0.98ath-miR159a 0.70 1.74 9.17 1.12 5.95 0.19 499.99 167.53 3.69 0.61 3.68ath-miR167c 0.27 0.57 40.05 1.91 13.41 2.57 13523.59 8992.73 55.09 0.3862.65 ath-miR319 0.00 0.00 0.24 0.05 0.11 0.01 375.35 61.39 0.43 0.051.28 ath-miR399a 0.00 0.01 0.86 0.03 0.29 0.00 208.27 39.40 0.05 0.008.12 ath-miR159b 0.24 0.96 3.19 0.44 2.13 0.10 217.23 60.24 1.34 0.212.73 ath-miR167d 0.09 0.12 6.22 0.18 8.87 3.08 74431.14 37597.33 103.190.03 1.84 ath-miR319c 0.00 0.00 0.03 0.00 0.01 0.00 10.09 1.85 0.01 0.000.06 ath-miR399b 0.00 0.01 0.84 0.02 0.33 0.00 116.03 17.61 0.02 0.0020.56 ath-miR159c 0.02 0.07 0.90 0.03 0.17 0.01 30.71 6.50 0.21 0.060.10 ath-miR168 0.08 0.08 8.11 0.66 1.02 0.20 633.43 335.35 3.01 0.033.05 ath-miR393a 0.00 0.00 0.85 0.01 0.09 0.01 234.90 58.76 0.10 0.000.14 ath-miR399d 0.00 0.00 0.07 0.00 0.02 0.00 21.85 3.45 0.01 0.00 0.95ath-miR160 0.63 1.68 10.22 1.96 2.24 0.16 932.78 272.42 2.71 0.26 14.36ath-miR169 0.01 0.02 1.88 0.05 1.73 0.02 222.26 172.16 0.39 0.01 0.31ath-miR394a 0.04 0.11 11.24 1.16 6.91 0.10 570.12 341.75 1.23 0.18 7.88ath-miR399e 0.00 0.00 0.00 0.00 0.00 0.00 0.33 0.10 0.00 0.00 0.04ath-miR161 0.01 0.00 0.01 0.02 0.00 0.00 1.44 0.72 0.01 0.00 0.00ath-miR169b 0.01 0.00 2.18 0.13 1.04 0.01 165.48 81.61 0.44 0.01 0.73ath-miR395a 0.00 0.00 0.12 0.04 3.01 0.00 12.26 2.13 0.03 0.00 0.01ath-miR399f 0.00 0.00 0.05 0.00 0.03 0.00 21.54 3.27 0.00 0.00 0.94ath-miR162 0.00 0.02 1.14 0.04 0.36 0.01 23.51 9.79 0.12 0.00 0.11ath-miR169d 0.01 0.01 3.58 0.16 1.97 0.02 309.21 167.12 0.77 0.02 0.82ath-miR395b 0.04 0.03 0.07 0.04 1.10 0.02 7.49 1.54 0.05 0.00 0.00ath-miR163 0.00 0.00 0.00 0.00 0.00 0.00 0.03 0.02 0.00 0.00 0.00ath-miR169h 0.01 0.00 1.49 0.21 13.93 0.00 23.86 37.05 0.06 0.00 2.48ath-miR396a 0.06 0.12 15.62 0.37 21.77 0.57 33028.58 9829.62 6.79 0.1466.63 R2, R3, R4, and R5 represent different reproductive stages duringkernel development: R2 stage (blister, 10-14 days after silking); R3stage (milk, 18-22 days after silking); R4 stage (dough, 24-28 daysafter silking); R5 stage (dent, 35-42 days after silking). Callusrepresents embryogenic calli that were induced during regenerationprocess.

For example, miR319 and miR398a are expressed in kernel and callus(embryogenic calli). MiR167d and miR167 family members are highlyexpressed in different developmental stages of kernel. MiR172 hasrelatively high expression in root, tassel and kernel.

Example 11: Construction of Binary Vectors with miRNA Tags

Based on miRNA expression profiling, several binary vectors wereconstructed in such way that a short nucleotide sequence nearlycomplementary to miRNA was incorporated into 3′ UTR of dsRed. RPR40 (SEQID NO: 216) was a negative control in which dsRed expression was underthe control of ScBV promoter and NOS terminator. Two unique restrictionenzyme sites, Sac I and Ava II, located between translation stop codon‘TAG’ of dsRed and NOS terminator were used to insert a short nucleotidesequence to create RPR41, RPR42, RPR43, RPR44 and RPR45, respectively.Each short nucleotide sequence was determined by analyzing the region ofmRNA potentially targeted by miRNA. For example, maize glossy 15 istargeted by miR172 in the region 5′ CTGCAGCATCATCAGGATTCC 3′ (i.e. miRNAtag) which is nearly complementary to miR172, 5′ AGAAUCUUGAUGAUGCUGCAC3′. A short oligo (SEQ ID NO: 220) containing miR172 target region plus5 nt up and downstream, and Sac I and Ava II sites was chemicallysynthesized. This short oligo was then subcloned into RPR40 to createRPR42.

TABLE 17 Vectors and miRNA tags used for leakiness control Specificsequence Predicted containing MiRNA DsRed2 Construct ID MiRNA tag miRNAtag Expression expression RPR40 None N/A N/A Everywhere (SEQ ID NO: 216)RPR41 MiR319 SEQ ID Kernel and Everywhere but NO: 221 callus weak or noin kernel and callus RPR42 MiR172 SEQ ID Root, tassel, Everywhere butNO: 220 low in kernel weak or no in root and tassel RPR43 MiR396a SEQ IDKernel and Everywhere but NO: 222 callus weak or no in kernel and callusRPR44 MiR398a SEQ ID Kernel Everywhere but NO: 223 weak or no in kernelRPR45 MiR167d SEQ ID High in kernel, Everywhere but NO: 224 callus weakor no in kernel and callus

Example 12: Gene Silencing of miRNA Tagged DsRed2 in Maize Callus

12.1 Generation of Transgenic Calli

Immature maize embryos were transformed with Agrobacterium containingeither plasmids RPR40, RPR41, or RPR42. The transformation and selectionprocedures are modified from Ishida et al. (1996, Nature Biotech14:745-749). Immature embryos were excised and placed into infectionmedia. Infection media was removed and replaced with a suspension ofAgrobacterium pre-induced for 1-4 hours in infection media containing200 μM acetosyringone. Agrobacterium and embryos remained in liquid for30 minutes for infection. Following infection, Agrobacterium solutionwas removed and embryos placed on co-culture media (modified from Ishidawith the addition of 150 mg/L L-cysteine). Co-culture was allowed tooccur for 2-3 days. Following co-culture, embryos were placed on arecovery media containing antibiotics to inhibit Agrobacterium growthfor 7-10 days. Embryos that formed callus were placed on a selectionmedia capable of suppressing growth of non-transformed tissue.

12.2 Identification of Transgenic Calli and Copy Number Analysis

The transgene copy number in maize calli transformed with RPR40 andRPR41 was determined by TaqMan analysis (Ingham et al., 2001,Biotechniques 31:132-4, 136-40). The TaqMan probe was chosen to targetNOS terminator, which is located downstream of the DsRed2s as a commonregion in these three constructs. Only the transgenic maize calli wereused for the following expression analysis for DsRed2.

12.3 Isolation of RNA from Transgenic Maize Callus

Callus tissues were ground with a mortar and pestle in liquid nitrogenfollowed by addition of 600 μL of lysis/binding solution (mirVana miRNAIsolation Kit, Ambion, Inc. Austin, Tex.) in order to extract total RNAbased on the mirVana total RNA isolation protocol. The isolated RNA wasDNase treated with DNA-free (Ambion, Inc) following the manufacture'sprotocol.

12.4 DsRed RNA Quantitation

DsRed2 and the endogenous maize GpC1 first strand cDNAs were synthesizedfrom RNA isolated from RPR40 and RPR41 maize transgenic calli. RNA wasreversed transcribed with the ImProm-II Reverse Transcription System(Promega, Madison, Wis.) using DsRed and GpC1 specific primers andfollowing the manufacture's protocol. The relative levels of DsRed2 RNAfrom the RPR40 and RPR41 transgenic calli were determined byquantitative Taqman PCR using probes specific to DsRed2 and GpC1. Firststrand cDNA synthesized from callus total RNA was used as template. TheTaqMan assay was performed essentially as for copy number analysis. Tocompare the relative amounts of DsRed2 RNA between calli, the data werefirst normalized to the internal GpC1 endogenous control. Quantitationof DsRed2 RNA was repeated 3 times for each RNA sample.

12.5 Fluorescence is Reduced in Maize Calli Expressing DsRed2 Taggedwith the 319 and 172 miRNA Binding Sites

Putative transgenic calli containing plasmids RPR40, RPR41, and RPR42were examined for DsRed2 fluorescence under a microscope (Zeiss StemiSV11) equipped with UV and rhodamine filter. The intensity offluorescence was recorded as high, medium, low and none (see Table 18).

TABLE 18 DsRed2 fluorescent expression of transgenic calli Total numberof Construct ID High Medium Low None calli examined RPR40 17 2 11 2 32RPR41 0 3 13 17 33 RPR42 14 8 8 17 47

12.5 miRNA Tagged DsRed2 RNA is Significantly Reduced in Maize Calli

Each cluster of calli analyzed was confirmed to be transgenic byquantitative TaqMan PCR. This assay also provided a copy number ofintegrated DsRed2 constructs in each cluster of calli. To compare therelative levels of DsRed2 RNA in the RPR40 and RPR41 calli populations,RNA was isolated from individual transgenic positive calli and theamount of DsRed2 RNA determined by quantitative TaqMan analysis. TheDsRed2 RNA is greatly reduced in RPR41 transgenic calli compared toRPR40 calli (Table 19). The difference in DsRed2 RNA levels between theRPR40 and RPR41 calli populations is significant with a p value of0.0026.

TABLE 19 DsRed2 mRNA expression from transgenic calli Quantitation ofDsRed2 copy RNA (3 repetitions) callus # plasmid miRNA tag number A B C1 RPR40 none 1 25.2 35.1 30.1 2 RPR40 none 1 46.8 43.0 48.3 3 RPR40 none1 26.8 22.2 23.1 4 RPR40 none 1 50.6 30.6 38.5 5 RPR40 none 1 37.7 46.039.7 6 RPR40 none 1 7.8 8.5 11.1 7 RPR40 none 1 11.6 56.0 17.4 8 RPR41mi319 3 8.9 8.6 9.6 9 RPR41 mi320 2 8.0 7.6 8.0 10 RPR41 mi321 1 1.1 1.01.4 11 RPR41 mi322 1 6.2 10.1 7.3 12 RPR41 mi323 1 11.5 12.5 14.3

We claim:
 1. An expression construct comprising a promoter sequencefunctional in a monocotyledonous plant and functionally linked thereto achimeric nucleotide sequence to be expressed, wherein said chimericnucleotide sequence comprises: i) at least one sequence comprising anopen reading frame encoding a protein that is capable of conferring anagronomically valuable trait in a monocotyledonous plant, wherein theagronomically valuable trait is selected from the group consisting ofdisease resistance, pest resistance, herbicide resistance, sterility,grain characteristics, vigor, time to harvest, enhanced nutrientcontent, novel growth patterns, flavors or colors, and salt, heat,drought, and/or cold tolerance, and ii) at least one sequence having atleast 95% sequence complementarity to a tissue-specifically expressedmicroRNA sequence naturally occurring in said monocotyledonous plant,wherein the microRNA is a plant microRNA comprising: a) the nucleotidesequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234,235, 236, 237, 238, 239, 240, 241, 242, 243, 245, 245, 246, 247, 248,249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262,263, 264, 265, or 266; or b) a nucleotide sequence having at least 95%sequence identity to the nucleotide sequence of SEQ ID NO: 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 225, 226, 227,228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241,242, 243, 245, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255,256, 257, 258, 259, 260, 261, 262, 263, 264, 265, or 266; wherein the atleast one sequence of i) and the at least one sequence of ii) areheterologous to each other.
 2. The expression construct of claim 1,wherein said promoter is selected from the group consisting ofconstitutive promoters, tissue-preferential promoters, developmentalpromoters, and inducible promoters.
 3. The expression construct of claim1, wherein mismatches between the at least one sequence of ii) and themicroRNA sequence naturally occurring in the plant are in the regioncorresponding to the 3′-region of said microRNA sequence.
 4. Theexpression construct of claim 1, wherein the microRNA is tissue-specificexpressed, spatially-regulated, developmental regulated, and/orregulated by biotic or abiotic stress factors.
 5. The expressionconstruct of claim 1, wherein said expression construct is in a plasmid.6. The expression construct of claim 1, wherein the microRNA has anexpression profile in the plant selected from the group consisting of:a) expression in any tissue but no expression in seed; b) expression inseeds but not in other tissues; c) drought or other abioticstress-induced expression; d) plant pathogen-induced expression; and e)chemical induced expression.
 7. The expression construct of claim 1,wherein the at least one sequence of ii) is positioned in a location ofthe nucleotide sequence to be expressed corresponding to the5′-untranslated region or the 3′-untranslated region of said nucleotidesequence.
 8. An expression vector comprising the expression construct ofclaim
 1. 9. A transformed cell or a non-human organism comprising theexpression construct of claim
 1. 10. The transformed cell or non-humanorganism of claim 9, wherein said expression construct is inserted intoits genome.
 11. The transformed cell or non-human organism of claim 9,wherein said cell or organism is selected from the group consisting ofmammalian, bacterial, fungal, nematode, and plant.
 12. A transgenicmonocot plant, plant cell or plant part comprising: a) the expressionconstruct of claim 1; or b) a vector comprising said expressionconstruct of a).
 13. A pharmaceutical preparation comprising: a) theexpression construct of claim 1; or b) a vector comprising saidexpression construct of a).
 14. The expression construct of claim 1,wherein the plant microRNA comprises a nucleotide sequence having atleast 95% sequence identity to the nucleotide sequence of SEQ ID NO: 22.15. The expression construct of claim 1, wherein the plant microRNAcomprises the nucleotide sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 225, 226, 227, 228, 229,230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243,245, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257,258, 259, 260, 261, 262, 263, 264, 265, or
 266. 16. The expressionconstruct of claim 1, wherein the microRNA comprises a nucleotidesequence identical to SEQ ID NO: 22.